Polypeptide Display Libraries and Methods of Making and Using Thereof

ABSTRACT

Disclosed herein are expression vectors which display a passenger polypeptide on the outer surface of a biological entity. As disclosed herein the displayed passenger polypeptide is capable of interacting or binding with a given ligand. Also disclosed are methods of making and using the expression vectors. N/C terminal fusion expression vectors and methods of making and using are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/920,244, filed 18 Aug. 2004, allowed, which claims the benefit ofU.S. Provisional Patent Application No. 60/495,698, filed 18 Aug. 2003,listing Patrick S. Daugherty, Paul H. Bessette, and Jeffrey Rice, asjoint inventors, both of which are herein incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to bacterial polypeptide displaylibraries and methods of making and using thereof.

2. Description of the Related Art

Polypeptide display technologies have substantially impacted basic andapplied research applications ranging from drug discovery to materialssynthesis. See Clackson, T. and J. A. Wells (1994) Trends In Biotech.12(5):173-184; and Shusta, E. V., et al. (1999) Curr. Opin. Biotechnol.10(2):117-122; and Kodadek, T., (2001) Chem. Biol. 8(2):105-158; Lee, S.W., et al. (2002) Science 296 (5569):892-859; and Nixon, A. E. (2002)Curr. Pharm. Biotechnol. 3(1):1-12. The strength of these methodsderives from the ability to generate libraries containing billions ofdiverse molecules using the biosynthetic machinery of the cell, andsubsequently, to identify rare desired polypeptides using selection orhigh-throughput screening methods. Display libraries have been appliedextensively to isolate and engineer peptides and antibodies formolecular recognition applications. In particular, display of peptideson the surface of filamentous bacteriophage, or phage display, hasproven a versatile and effective methodology for the isolation ofpeptide ligands binding to a diverse range of targets. See Scott, J. K.and G. P. Smith (1990) Science 249(4967):386-904; Norris, J. D., et al.(1999) Science 285(5428):744-765; Arap, W., et al. (1998) Science279(5349):377-806; and Whaley, S. R., et al. (2000) Nature405(6787):665-668.

Polypeptide display systems include mRNA and ribosome display,eukaryotic virus display, and bacterial and yeast cell surface display.See Wilson, D. S., et al. 2001 PNAS USA 98(7):3750-3511; Muller, O. J.,et al. (2003) Nat. Biotechnol. 3:312; Bupp, K. and M. J. Roth (2002)Mol. Ther. 5(3):329-3513; Georgiou, G., et al., (1997) Nat. Biotechnol.15(1):29-3414; and Boder, E. T. and K. D. Wittrup (1997) Nature Biotech.15(6):553-557. Surface display methods are attractive since they enableapplication of fluorescence-activated cell sorting (FACS) for libraryanalysis and screening See Daugherty, P. S., et al. (2000) J. Immunol.Methods 243(1-2):211-2716; Georgiou, G. (2000) Adv. Protein Chem.55:293-315; Daugherty, P. S., et al. (2000) PNAS USA 97(5):2029-3418;Olsen, M. J., et al. (2003) Methods Mol. Biol. 230:329-342; and Boder,E. T. et al. (2000) PNAS USA 97(20):10701-10705.

Phage display involves the localization of peptides as terminal fusionsto the coat proteins, e.g., pIII, pIIV of bacteriophage particles. SeeScott, J. K. and G. P. Smith (1990) Science 249(4967):386-390; andLowman, H. B., et al. (1991) Biochem. 30(45):10832-10838. Generally,polypeptides with a specific function of binding are isolated byincubating with a target, washing away non-binding phage, eluting thebound phage, and then re-amplifying the phage population by infecting afresh culture of bacteria. Unfortunately, phage display presents a fewundesirable properties. See Zahn, G. (1999) Protein Eng.12(12):1031-1034. For example, phage display is limited to about a fewthousand copies of the displayed polypeptide per phage or less, therebyprecluding the use of sensitive fluorescence-activated cell sorting(FACS) methodologies for isolating the desired sequences. Phage are alsodifficult to elute or recover from an immobilized target ligand, therebyresulting in clonal loss. Phage display also requires an infection stepwherein viruses that do not bind and enter a cell are lost early in theprocess, thereby resulting in lower quality results overall, e.g.,affinity of isolated binding molecules. Further, phage displayselections are time consuming requiring typically about two to aboutthree weeks for the isolation of phage display polypeptides that bind agiven target.

Most notably, phage display requires that the investigator be familiarwith routine phage manipulation methods including infections, phageamplifications, tittering, phage ELISA, and others. Second, phagedisplay methods can lead to Darwinian outgrowth of particular clonesowing to their relative infectivity, assembly efficiency, and toxicityto the host cell. Third, the rate at which desired binding clones can beenriched is slowed by relatively low enrichment ratios.

Other display formats and methodologies include mRNA display, ribosomeor polysome display, eukaryotic virus display, and bacterial, yeast, andmammalian cell surface display. See Matthcakis, L. C., et al. (1994)PNAS USA 91(19): 9022-9026; Wilson, D. S., et al. (2001) PNAS USA98(7):3750-3755; Shusta, E. V., et al. (1999) Curr. Opin. Biotech.10(2):117-122; and Boder, E. T. and K. D. Wittrup (1997) Nature Biotech.15(6):553-557. A variety of alternative display technologies have beendeveloped and reported for display on the surface of a microorganism andpursued as a general strategy for isolating protein binding peptideswithout reported successes. See Maurer, J., et al. (1997) J. Bacteriol.179(3):794-804; Samuelson, P., et al. (1995) J. Bacteriol.177(6):1470-1476; Robert, A., et al. (1996) FEBS Letters 390(3):327-333; Stathopoulos, C., et al. (1996) Appl. Microbiol. & Biotech.45(1-2): 112-119; Georgiou, G., et al., (1996) Protein Engineering 9(2):239-247; Haddad, D., et al., (1995) FEMS Immunol. & Medical Microbiol.12(3-4):175-186; Pallesen, L., et al., (1995) Microbiol. 141(Pt 11):2839-2848, Xu, Z. and S. Y. Lee (1999) Appl. Environ. Microbiol.65(11):5142-5147; Wernerus, H. and S. Stahl (2002) FEMS Microbiol. Lett.212(1): 47-54; and Westerlund-Wikstrom, B. (2000) Int. J. Med.Microbiol. 290(3):223-230. Some of these prior art display systems havebeen tested for library screening without success for isolation of highaffinity protein binding peptides. See Brown, S. (1992) PNAS USA89(18):8651-8655; Lang, H., et al. (2000) Eur. J. Biochem.267(1):163-170; Klemm, P. and M. A. Schembri (2000) Int. J. Med.Microbiol. 290(3):215-221; Klemm, P. and M. A. Schembri (2000)Microbiol. 146(Pt 12):3025-3032; Kjaergaard, K., et al. (2000) Appl.Environ. Microbiol. 66(1):10-14; Schembri, M. A., (1999) FEMS Microbiol.Lett. 170(2):363-371; Benhar, I., et al. (2000) J. Mol. Biol.301(4):893-904; and Lang, H., et al. (2000) Adv. Exp. Med. Biol.485:133-136.

Prior art expression vectors for polypeptide display libraries usinghost cells suffer from a variety of problems. The problems of the priorart methods include (1) only small peptides may be expressed, (2) largelibraries cannot be selected, (3) the polypeptides are not expressed onthe outer membrane surface, but are instead expressed in the periplasmicspace between the inner and the outer membranes, (4) polypeptides thatare displayed on the outer membrane surface do not properly bind orinteract with large molecules and certain targets, and (5) analyzingexpression on fimbrial or flagella results in loss of some desiredpolypeptides due to mechanical shearing.

Protein display on the surface of bacterial cells holds the potential tosimplify and accelerate the process of ligand isolation sinceexperimental procedures with bacteria are efficient and screening can beperformed using FACS. See Daugherty, P. S., et al. (2000) J. Immunol.Methods 243(1-2):211-2720; Brown, S. (1992) PNAS USA 89(18):8651-8521;and Francisco, J. A., et al. (1993) PNAS USA 90(22):10444-10448;Taschncr, S., et al. (2002) Biochem. J. 367(Pt 2):393-402; Etz, H., etal. (2001) J. Bacteriol. 183(23):6924-6935; and Camaj, P., et al. (2001)Biol. Chem. 382(12):1669-1677. Though several different bacterialdisplay systems have been reported, their usefulness has been restrictedby technical limitations including accessibility on the cell surface,inability to display highly diverse sequences, and adverse effects oncell growth and viability. See Francisco, J. A., et al. (1993) PNAS USA90(22):10444-10822; Lu, Z., et al. (1995) Biotechnology (NY)13(4):366-7223; Klemm, P. and M. A. Schembri, (2000) Microbiology 146(Pt12):3025-3224; Christmann, A., et al. (1999) Protein Eng.12(9):797-80625; Lee, S. Y., et al. (2003) Trends Biotechnol.21(1):45-52; Lu, Z., et al. (1995) Biotechnology (NY) 13(4):366-7225;Lee, S. Y., et al. (2003) Trends Biotechnol. 21(1):45-5226; Camaj, P.,et al. (2001) Biol. Chem. 382(12):1669-1677; and Schembri, M. A., et al.(2000) Infect. Immun. 68(5):2638-2646.

Consequently, these techniques do not enable isolation of high affinitypeptide ligands. Additionally, these techniques do not provide peptideexposure on the cell surface suitable for binding to analytes includingantibodies, proteins, viruses, cells, macromolecules. Thus, thesedisplay formats are not compatible with certain isolation methods, sincethe peptides produced do not bind to large molecules and other surfaces,e.g., magnetic particles. The prior art process also reduces cellviability and alters membrane permeability, thereby reducing processefficiency. Thus far, routine isolation of high affinity peptide ligandsfor arbitrary protein targets has not been demonstrated. See Camaj, P.,et al., (2001) Biol. Chem. 382(12):1669-7727; and Tripp, B. C., et al.,(2001) Protein Eng. 14(5):367-377; Lang, H., et al. (2000) Eur. J.Biochem. 267(1):163-170; Lang, H., et al. (2000) Adv. Exp. Med. Biol.485:133-136; Klemm, P. and M. A. Schembri (2000) Int. J. Med. Microbiol.290(3): 215-221; Klemm, P. and M. A. Schembri (2000) Microbiol. 146(Pt12):3025-302; Kjaergaard, K., et al. (2000) Appl. Environ. Microbiol.66(1):10-14; Schembri, M. A., et al. (1999) FEMS Microbiol. Lett.170(2):363-371; Benhar, I., et al. (2000) Mol. Biol. 301(4):893-904;Kjaergaard, K., et al. (2001) Appl. Environ. Microbiol.67(12):5467-5473; and Lang, H., et al. (2000) Exp. Med. Biol.485:133-136.

Also, polypeptides in the prior art are most often displayed on cellsurfaces either as insertional fusions or “sandwich fusions” into outermembrane or extracellular appendage, e.g., fimbria, flagella proteins,or less frequently, as fusions to truncated or hybrid proteins thoughtto be localized on the cell surface. See Pallesen, L., et al. (1995)Microbiol. 141(Pt 11):2839-48; and Etz, H., et al. (2001) J. Bacteriol.183(23):6924-6935. Examples of the latter include the LppOmpA system andthe ice nucleation protein (InP). See Georgiou, G., et al. (1997) Nat.Biotechnol. 15(1):29-34. The outer membrane proteins OmpA, OmpC, OmpF,FhuA, and LamB, have enabled the display of polypeptides as relativeshort insertional fusions into OMP loops exposed on the extracellularside of the outer membrane. See Xu, Z. and S. Y. Lee (1999) Appl.Environ. Microbiol. 65(11):5142-5147; Taschner, S., et al. (2002)Biochem. J. 367(Pt 2):393-402.

However, the C and N-termini of these “carrier” proteins are notnaturally located on the cell surface which precludes the ability todisplay polypeptides as terminal fusions. As a result, proteins whichare not capable of folding in the insertional fusion context, when theirC and N termini are fused to the “carrier” protein sequence, as well asthose for which the C and N termini are physically separated in space,e.g., single chain Fv antibody fragments, cannot be displayedeffectively as insertions. Similarly, the restriction to the use ofinsertional fusions, interferes with the display of a large number ofproteins encoded by cDNA libraries on the cell surface.

Prior art methods have attempted to address the problems of insertionalfusion displays by truncating outer membrane protein sequences such thatthe resulting new termini might be displayed on the cell surface. SeeLee, et al. (2003) Trends in Biotech. 23(1):45-52; Georgiou, et al.(1997) Nat. Biotech. 15(1):29-34. These prior art approaches were usedto create the LppOmpA system which allows for the targeting of peptidesand polypeptides to the outer membrane of bacteria. See Francisco, etal. (1992) PNAS USA 89(7):2913. For example, expression vectors forwhich use LppOmpA′, araBAD promoter, chloramphenicol resistance, and ap15A origin (LppOmpA expression vector). See Daugherty et al. (1999)Protein Engineer. 12(7):613-621. The LppOmpA expression vector encodes afusion protein that results in a truncation of the OmpA protein at aminoacid residue 159. Unfortunately, the performance of LppOmpA expressionvector as a general process for isolating and expressing polypeptidesfrom large libraries is significantly restricted by i) the reducedstructural stability of the modified OmpA protein, ii) intolerance toexpression at high temperatures, iii) reduced viability, and iv) mostimportantly, its inability to display polypeptides on the cell surfacein a manner compatible with binding to large proteins withoutcompromising viability and/or growth rate See Christman, A. et al.,1999. Prot. Eng. 12 (9):797.

In addition, expression vectors in the prior art are problematic because(1) the polypeptides produced by the expression vectors are not capableof binding externally added proteins, cells, or surfaces to the hostcells, (2) the expression vectors does not allow surface presentation oflarge polypeptides, and (3) the expressed polypeptides are onlyexpressed in the periplasmic region (between the inner and outermembrane) and not on the outer surface of the host cell, and thereforeany expressed protein can only interact with small molecules that passthrough the outer membrane and into the periplasmic space. Theseproblems have prevented the application of this technology as a generalprocess for isolating high affinity binding polypeptides. See e.g.,Stathopoulos, C. (1996) Applied Microbiol. Biotech. 45 (1-2) 112.Earhart CF. (2000) Methods Enzymol. (326):506-16; Francisco, J. (1994)Annal. NY Acad. Sci. 745:372; and Bessette, P. H., et al. (2004) Prot.Eng. (In Press).

Thus, a need exists for a more robust display methodology which requiresminimal technical expertise, is less labor intensive, and speeds theprocess of ligand isolation from weeks to days as compared to the priorart methods.

SUMMARY OF THE INVENTION

The present invention relates to expression vectors for displayingpolypeptides on an outer surface of a biological entity within a carrierprotein loop.

In some embodiments, the present invention provides an expression vectorcapable of expressing and displaying a given passenger polypeptide on anouter surface of a biological entity within a carrier protein loop thatis capable of interacting with a given ligand.

In some embodiments, the carrier protein loop is opened resulting in anN-terminus exposed on the outer surface, a C-terminus exposed on theouter surface, or both. In some embodiments, the native C-terminus andthe native N-terminus are fused together via a peptide linker. In someembodiments, the N-terminus and the C-terminus exposed to the outersurface are accessible by the ligand. In some embodiments, the Cterminus of the passenger polypeptide is fused to the N terminus of thecarrier protein. In some embodiments, the N terminus of the passengerpolypeptide is fused to the C terminus of the carrier protein. In somepreferred embodiments, the carrier protein is OmpX.

In some embodiments, the carrier protein is a bacterial outer membraneprotein. In some preferred embodiments, the bacterial outer membraneprotein is OmpA or OmpX. In some preferred embodiments, the polypeptideis expressed in the first extracellular loop of OmpA. In some preferredembodiments, the polypeptide is expressed in the second extracellularloop of OmpX. In some preferred embodiments, the polypeptide isexpressed in the third extracellular loop of OmpX.

In some embodiments, the polypeptide is streptavidin or a T7 bindingpeptide.

In some embodiments, the biological entity is a bacterial cell, a yeastcell or a mammalian cell. In some preferred embodiments, the biologicalentity is a bacterial cell. In some preferred embodiments, the bacterialcell is Escherichia coli, Shigella sonnei, Shigella dysenteriae,Shigella flexneri, Salmonella typhimurium, Salmonella enterica,Enterobacter aerogenes, Serratia marcescens, Yersinia pestis, orKlebsiella pneumoniae.

In some embodiments, the expression vector further comprises a low copyorigin of replication, such as a p15A origin of replication.

In some embodiments, the expression vector further comprises abacteriocidal antibiotic resistance protein encoding gene. In someembodiments, the bacteriocidal antibiotic resistance protein encodinggene encodes chloramphenicol acetyltransferase.

In some embodiments, the expression vector further comprises at leastone SfiI endonuclease restriction enzyme site.

In some embodiments, the expression vector further comprises anarabinose araBAD E. coli operon promoter. In some embodiments,expression is induced with the addition of L-arabinose and stopped bythe removal of arabinose and the addition of glucose.

In some embodiments, the present invention provides a host cell whichcomprises an expression vector as provided herein.

In some embodiments, the present invention provides a method of making apolypeptide display library which comprises creating a plurality ofexpression vectors capable of expressing a plurality of polypeptidesaccording to that described herein and inducing expression.

In some embodiments, the present invention provides a polypeptideexpressed on the outer surface of a biological entity by inducingexpression of an expression vector described herein. In someembodiments, the polypeptide is expressed in the first extracellularloop of OmpA. In some embodiments, the polypeptide is expressed in thesecond extracellular loop of OmpX. In some embodiments, the polypeptideis expressed in the third extracellular loop of OmpX.

In some embodiments, the present invention provides a polypeptideexpressed on the outer surface of a biological entity by inducingexpression of an expression vector having a carrier protein loop openedand an N-terminus exposed on the outer surface, a C-terminus exposed onthe outer surface, or both exposed to the outer surface, as describedherein. In some embodiments, the polypeptide is expressed in OmpX.

In some embodiments, the present invention provides a polypeptidedisplay library which comprises a polypeptide expressed and displayed byan expression vector described herein.

In some embodiments, the present invention provides an assay method fordetecting, monitoring, or measuring a given ligand in a sample whichcomprises inducing an expression vector described herein to express thepolypeptide and then contacting the polypeptide with the sample andobserving whether the polypeptide interacts with the ligand.

In some embodiments, the carrier polypeptide of the expression vector ofthe present invention is encoded by a nucleic acid molecule whichcomprises at least one codon that encodes a given amino acid that isreplaced with a replacement codon which encodes an alternate amino acidthat is structurally similar to the given amino acid. In someembodiments, all the codons that encode the given amino acid arereplaced. In some embodiments, the biological entity incorporates atleast one non-canonical amino acid analog into the displayedpolypeptide. In some embodiments, the given amino acid is leucine. Insome embodiments, the alternate amino acid is valine, isoleucine, ortrifluorleucine.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed. The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitutepart of this specification, illustrate several embodiments of theinvention and together with the description serve to explain theprinciples of the invention.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1 shows disulphide loops identified de novo, enabling high affinitybinding to target proteins. The first sequence in the upper left box isSEQ ID NO:186, the second sequence in the upper left box is SEQ IDNO:187, the first sequence in the upper right box is SEQ ID NO:188, thesecond sequence in the upper right box is SEQ ID NO:189, the firstsequence in the lower left box is SEQ ID NO:190, the second sequence inthe lower left box is SEQ ID NO:191, the first sequence in the lowerright box is SEQ ID NO:192, the second sequence in the lower right boxis SEQ ID NO:193.

FIG. 2 is an illustration of the location of OmpA loop 1 insertionsmediating high level display of oligopeptides, and location of SfiIcloning sites enabling high efficiency cloning.

FIG. 3A shows a schematic representation of display of peptides on thesurface of E. coli using insertions into the first extracellular loop(L1) of outer membrane (OM) protein A (OmpA). LPS=lipopolysaccharide.

FIG. 3B shows a histogram of flow cytometric analysis of clonalpopulation of cells containing plasmid pB33OmpA overexpressing OmpAwithout any insertions. Cells were induced for 2 hours and labeled with10 nM biotinylated-anti-T7•tag mAb and phycoerythrin-conjugatedstreptavidin.

FIG. 3C shows cells containing the plasmid pB330T1 displaying the T7•tagpeptide in OmpA loop 1, induced and labeled as in Figure AB.

FIG. 4 shows lack of growth inhibition for cells displaying peptides(MC1061/pBAD33L1) as compared to cells not displaying peptides(MC1061/pBAD33OmpA), and cells displaying peptides usingMC1061/pBAD18OmpAL1).

FIG. 5 shows maintenance of library diversity through 80 doublingsindicating that library can be expanded indefinitely for reuse.

FIG. 6A shows two permissive sites for polypeptide display in OmpXidentified by multiple sequence alignment. The first sequence is SEQ IDNO:194 and the second sequence is SEQ ID NO:195. Sites are suitablesince they (1) are located more than about 1 nM from the cell surface,(2) they are non-conserved across different species, (3) they exhibitconformational flexibility, and (4) they are located in a relativelysmall monomeric Omp protein.

FIG. 6B shows bacterial display libraries as N-terminal fusions to acircularly permuted variant of OmpX.

FIG. 7A shows the fluorescence intensity of E. coli cells (MC1061)containing expression plasmids: pBAD33CPX (expressing N-terminal CPXwithout any passenger protein), pBAD18Grn (expressing GFP from a ColE1origin), pBAD33Grn expressing GFP from a plasmid with a p15A origin,pBAD18GCS co-expressing alajGFP (G) and SA-1 (Table 2) within theN-terminal CPX scaffold, and pBAD33GCS expressing AlajGFP (G) and SA-1within the N-terminal CPX scaffold, as measured using flow cytometry.

FIG. 7B shows fluorescence microscopy analysis of tumor cells incubatedwith bacterial cells MC1061/pBAD33OmpA, which overexpress OmpA without atargeting peptide. Bacteria co-express an autofluorescent protein (e.g.alajGFP) internally, and a selected tumor binding/invading peptideexternally.

FIG. 7C shows fluorescence microscopy analysis of bacterial cells thattarget human breast cancer cells. MC1061 bacteria express anautofluorescent protein internally (e.g. AlajGFP), and a selected tumorbinding/invading peptide (YCLSYSNGRFFHCPA (SEQ ID NO:311)) externallyfrom plasmid pBAD33OmpA15.

FIG. 8A shows enrichment of C-reactive protein binding peptides asmeasured using flow cytometry. Induced cells were labeled with 10 nMbiotin-CRP and 6 nM SAPE in an unselected library population.

FIG. 8B shows enrichment of C-reactive protein binding peptides asmeasured using flow cytometry. Induced cells were labeled with 10 nMbiotin-CRP and 6 nM SAPE following two rounds of magnetic selection.

FIG. 8C shows enrichment of C-reactive protein binding peptides asmeasured using flow cytometry. Induced cells were labeled with 10 nMbiotin-CRP and 6 nM SAPE following two rounds of magnetic selection andone round of FACS.

FIG. 9 shows representative sequences of surface displayed high-affinityanti-T7tag mAb binding peptides isolated using magnetic selection andFACS with differing target ligand concentrations. Two rounds of MACSwere performed at 10 nM antibody concentration, followed by FACS using33 pM antibody. Bold residues indicate positions of identity with thewild-type T7•tag epitope, shown at the bottom, against which theantibody was raised. From top to bottom the sequence identifiers are SEQID NOs:196-208. The sequence identifier for the wild type sequence atthe bottom is SEQ ID NO:312.

FIG. 10A is a measurement of the binding affinities of cell surfacedisplayed streptavidin binding peptides using flow cytometry and biotinas a competitor as described herein.

FIG. 10B is a measurement of the binding affinities of cell surfacedisplayed streptavidin binding peptides using flow cytometry and biotinas a competitor as described herein. Determination of dissociation rateconstants (k_(diss)) of cell surface displayed peptides. Peptidesequences of clones SA-1 and SA-7 are listed in Table 2. Clone HPQcontains the sequence SAECHPQGPPCIEGR (SEQ ID NO:209) inserted into OmpAloop 1 for comparison.

FIG. 11 shows antibody epitope mapping of the antiT7tag antibody.Concentrations indicated are those used for screening using FACS. Thesequence identifiers for the top left box are: SEQ ID NO:210, SEQ IDNO:211, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214; for the topright box are: SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ IDNO:218, and SEQ ID NO:219; for the middle left box are: SEQ ID NO:220,SEQ ID NO:221, and SEQ ID NO:222; for the middle right box are: SEQ IDNO:223, SEQ ID NO:224, and SEQ ID NO:225; and for the bottom box: SEQ IDNO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, and SEQ ID NO:230and the w.t. is SEQ ID NO:231.

FIGS. 12A and 12B show the measurement of the dissociation rate ofstreptavidin binding peptide SA-1 grafted into a loop of YFP, on alinear plot (FIG. 12A) or on a semi-log plot (FIG. 12B). Flow cytometrywas used to measure the fluorescence of YFP bound to streptavidin coated1 μm beads after the addition of biotin as a competitor.

FIG. 13 shows equilibrium dissociation constants for streptavidinbinding sequences measured using flow cytometry. The sequenceidentifiers from top to bottom are SEQ ID NO:232, SEQ ID NO:233, and SEQID NO:234.

FIG. 14 shows an example of terminal fusion display using atopologically permuted Omp for polypeptide display exemplified usingOmpX. Using PCR methods familiar to one skilled in the art, a rearrangedgene sequence is assembled such that the order of the Omp polypeptidesequence is as shown in lower box in order to achieve N-terminalpolypeptide display within a surface exposed loop.

FIG. 15A shows flow cytometric analysis of control E. coli cellsoverexpressing OmpX. Cells were grown in LB growth medium, washed 1×,incubated with anti-T7tag monoclonal antibody, washed again, andincubated with 10 nM streptavidin phycoerythrin, and analyzed using flowcytometry.

FIG. 15B shows a two-parameter plot of Green vs. Red fluorescence of theidentical sample from 15A.

FIG. 15C shows E. coli displaying a T7tag peptide epitope recognized bya monoclonal antibody (MC1061/pCPX-T7). Cells were grown in liquidgrowth medium, washed 1× and incubated with anti-T7tag monoclonalantibody, washed again, and incubated with 10 nM streptavidinphycoerythrin, and analyzed using flow cytometry.

FIG. 15D shows a two-parameter plot of Green vs. Red fluorescence of theidentical sample from 15C.

FIG. 16 shows display of disulfide constrained peptides binding tostreptavidin (SA-1 pep), or non-constrained peptides binding to theanti-T7 epitope antibody (T7 pep) on the surface of E. coli usingrearranged OmpX display vector (CPX) resulting in either N terminaldisplay (N-CPX) or C-terminal display (C-CPX) of the passengerpolypeptide. Primary label concentration is the concentration of eitherstreptavidin-phycoerythrin, or anti-T7 monoclonal antibody used forfluorescent labeling.

FIG. 17 shows consensus sequences for streptavidin binding peptidesisolated from a fully random library displayed in OmpA loop. Thesequence identifiers from top to bottom are SEQ ID NOs:235-245.

FIG. 18 shows screening of intrinsically fluorescent bacterial displaypeptide libraries for tumor cell recognition using flow cytometry.

FIG. 19 shows CRP binding peptides possessing two distinct consensussequences. The sequence identifiers from top to bottom are SEQ IDNOs:246-256.

FIG. 20 shows peptide sequences isolated from a 15-mer library in OmpAbinding to ZR-75-1 human breast cancer tumor cells. The sequenceidentifiers from top to bottom are SEQ ID NOs:257-266.

FIG. 21 shows flow cytometric analysis of the OmpA 15mer library priorto selection (Unsorted Library) and populations resulting from one ortwo rounds of magnetic selection (MACS) for binding to a T7tag antibody.

FIG. 22 shows enrichment of CRP binding peptides by magnetic selection,as measured by flow cytometry.

FIG. 23 shows enrichment of tumor binding and internalizing bacteriausing FACS.

FIG. 24 shows dissociation rate constants of streptavidin bindingpeptides display on E. coli, measured by flow cytometry.

FIG. 25A shows streptavidin binding peptides selected from a doubleconstrained library, (XCCX₄CX₇CX) comprising about 1×10⁹ unique clonesdisplayed in loop 2 of OmpX. The sequence identifiers from top to bottomare SEQ ID NOs:267-275.

FIG. 25B shows streptavidin binding peptides selected from a X₄CX₃CX₄library displayed in loop 2 of OmpX. The sequence identifiers from topto bottom are SEQ ID NOs:276-284.

FIG. 26 shows HIV-1 gp120 binding peptides isolated using two cycles ofMACS, and one cycle of FACS. The sequence identifiers from top to bottomare SEQ ID NOs:285-297.

FIG. 27 shows the order and genetic elements required for C-terminaldisplay of the T7 peptide epitope in Loop 2 of OmpX T7 beginning withresidue 97 and ending with 95 (P96 deleted).

FIG. 28 shows an example methodology for construction of N-terminalfusion display using circular permutation, and loop opening between OmpXresidues 53/54. The displayed polypeptide is fused to residue 95, andthe leader peptide is genetically fused upstream to aa 97. In FIG. 28,the oligonucleotide primers represented by #1, #2, #4, and #5 are asfollows:

Primer (5′->3′) 1: Length 60 Melting Tm 48 Sense strand (SEQ ID NO: 131)ttcgagctcggtacctttgaggtggttatgaaaaaaattgcatgtctttcagcactggcc Primer(5′->3′) 2: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 132)tttcagcagtggccgcagttctggctttcaccgcaggtacttccgtagctatggcgagca Primer(5′->3′) 4: Length 60 Melting Tm 50 Sense strand (SEQ ID NO: 134)cggaggatctggtgactacaacaaaaaccagtactacggcatcactgctggtccggctta Primer(5′->3′) 5: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 135)gctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggt.

FIG. 29 shows primers used for the construction of N-terminal OmpXdisplay vector. Primers are as follows:

Primer 1 = Sense (SEQ ID NO: 298)ttcgagctcggtacctttgaggtggttatgaaaaaaattg (PD515) Primer 2 = Anti-Sense(SEQ ID NO: 299)ctggcctccacccatctgctggccgccggtcatgctcgccatagtagaagtcgcagctac Primer 3= Sense (SEQ ID NO: 300)ggccagcagatgggtggaggccagtctggccagtctggtgactacaacaaaaaccagtac Primer 4= Anti-Sense (SEQ ID NO: 301)cagtagaagtcgctccgcttcctccgaagcggtaaccaacaccgg Primer 5 = Sense (SEQ IDNO: 302) ggaggaagcggagcgacttctactgtaactggcggttacgcacag Primer 6= Anti-Sense (SEQ ID NO: 303)aaaacagccaagcttggccaccttggccttattagcttgcagtacggcttttctcg.

FIG. 30 shows the arrangement of OmpX fragments needed to enableC-terminal display of a passenger polypeptide. Oligonucleotide primersneeded to amplify and assemble the OmpX fragments resulting inC-terminal display are pictorially represented along with the resultingDNA products from application of the polymerase chain reaction. In FIG.30, the oligonucleotide primers represented by #1, #2, #4, and #5 are asdefined follows:

Primer (5′→3′) 1: Length 45 Melting Tm 49 Sense strand: (SEQ ID NO: 154)ttcgagctcggtacctttgaggtggttatgaaaaaaattgcatgt Primer (5′→3′) 2: Length57 Melting Tm 48 Antisense strand: (SEQ ID NO: 155)gcggtgaaagccagaactgcggccagtgctgaaagacatgcaattttttt cataacc Primer(5′→3′) 4: Length 57 Melting Tm 49 Antisense strand: (SEQ ID NO: 157)ttttccatcgggttgaactgcagacccgcaccgtaggagaaaccgtagtc gctggtg Primer(5′→3′) 5: Length 57 Melting Tm 48 Sense strand: (SEQ ID NO: 158)ttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccg tattcgt.

FIG. 31 shows the annealing locations of oligonucleotide primers thatcan be used to construct, via overlap PCR, a C-terminal display vectorusing OmpX. The primers are:

Primer 1 = Sense (SEQ ID NO: 304)ttcgagctcggtacctttgaggtggttatgaaaaaaattg (PD515); Primer 2 = Anti-Sense(SEQ ID NO: 305) ctggcctccacccatctgctcggccgccggtcatgctcgccatagtagaagtcgcagctac; Primer 3 = Sense (SEQ ID NO: 306)gccagcagatgggtggaggccagtctggccagtctggtgactacaacaaa aaccagtac; Primer 4= Anti-Sense (SEQ ID NO: 307)cagtagaagtcgctccgcttcctccgaagcggtaaccaacaccgg; Primer 5 = Sense (SEQ IDNO: 308) ggaggaagcggagcgacttctactgtaactggcggttacgcacag; Primer 6= Anti-Sense (SEQ ID NO: 309)tgctggccgccggtcatgctcgccatctggccagactggcctccgtattc agtggtctgg.

FIG. 32 is substantively duplicate of FIG. 31 and the primers are thesame as in FIG. 31.

FIG. 33 shows the display of polypeptide enzyme substrates using anN-terminal fusion display vector (N-CPX) for the selection,identification, and engineering of enzyme protease and peptidesubstrates, displaying the substrate and internally expressing a greefluorescent protein are green and red fluorescent. Cells treated with agiven protease which cleaves the surface substrate loose redfluorescence but remain green fluorescent. Isolation of fluorescentgreen (not red) cells allows the identification of substrates that canbe cleaved or lysed by a given protease.

FIG. 34 shows flow cytometric analysis of cells displaying of peptidesin an Omp encoding gene modified to possess no leucine codons, thusenabling display of peptides that incorporate a variety of syntheticleucine analogs. Display of a non-canonical amino acid is exemplifiedusing trifluoroleucine (Tfl) and OmpX. This figure also shows acomparison of the level of display of the T7tag peptide (that does notcontain any leucine residues) using either unmodified wild-type (top) orNoLeu-OmpX (bottom) scaffolds in the presence of (left) 19 amino acids(deficient in Leu), (middle) 19 amino acids+trifluoroleucine (No Leu),or (right) 20 standard amino acids. These were labeled with ananti-T7tag biotinylated antibody, washed once in PBS, and labeled withstreptavidin-phycoerythrin (Molecular Probes, Eugene, Oreg.). Theincrease in green fluorescence for the 19+Tfl samples (middle) fromabout 5.6 to about 63.7 allows for the screening of bacterial displaylibraries for peptides that incorporate Tfl.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an expression vector that expressesefficiently on the outer surface of a replicable biological entity agiven polypeptide, a “passenger” polypeptide, linked to a surfacelocalized polypeptide, herein referred to as a “carrier” polypeptide,that is otherwise deficient in a surface accessible C or N terminus.

As used herein, a “replicable biological entity” refers toself-replicating biological cells, including bacterial, yeast,protozoal, and mammalian cells, and various viruses capable of infectingthese cells known in the art, and the like.

As used herein, the terms “protein”, “polypeptide” and “peptide” areused interchangeably to refer to two or more amino acids linkedtogether.

Polypeptides presented according to the present invention (1) alleviatedisruption of the energetic structural stability of the carrierpolypeptide thus allowing presentation the preferred number of copies ofthe passenger polypeptide without a loss of viability, (2) are capableof interacting physically with arbitrary compositions of matter(biological or non-biological), and (3) exhibit a biological activity(e.g., affinity, specificity, catalysis, assembly etc.) substantiallysimilar to the corresponding free polypeptide in solution. In otherwords, the displayed polypeptide interacts with or binds a given targetmolecule in a manner that is substantially similar to that when thepolypeptide is in its native environment and not attached to thebiological entity.

As used herein, a “fusion protein” refers to the expression product oftwo or more nucleic acid molecules that are not natively expressedtogether as one expression product. For example, a native protein Xcomprising subunit A and subunit B, which are not natively expressedtogether as one expression product, is not a fusion protein. However,recombinant DNA methods known in the art may be used to express subunitsA and B together as one expression product to yield a fusion proteincomprising subunit A fused to subunit B. A fusion protein may compriseamino acid sequences that are heterologous, e.g., not of the sameorigin, not of the same protein family, not functionally similar, andthe like.

The polypeptides expressed and displayed according to the presentinvention may be large polypeptides yet still retain the ability to bindor interact with given ligands in a manner similar to the nativepolypeptide or the polypeptide in solution. As provided herein, theexpression vectors of the present invention use utilize a low copyorigin of replication and a regulatable promoter in order to minimizethe metabolic burden of the biological entity and the clonalrepresentation of the polypeptide library is not affected by growthcompetition during library propagation. The expression vectors of thepresent invention utilize a antibacterial resistance gene to abacteriocidal antibiotic which prevents plasmid loss and outgrowth ofcells resistant to the antibiotic. Additionally, the expression vectorsof the present invention lack a dual system, such as β-lactamase, whichresults in a smaller expression vector which imposes a smaller burden oncell growth and improves library screening. The expression vectors ofthe present invention also utilize a SfiI restriction site which allowsdigestion by a particular enzyme to generate overhangs that cannot reactwith incorrect DNA substrates.

As used herein, a “ligand” refers to a molecule(s) that binds to anothermolecule(s), e.g., an antigen binding to an antibody, a hormone orneurotransmitter binding to a receptor, or a substrate or allostericeffector binding to an enzyme and include natural and syntheticbiomolecules, such as proteins, polypeptides, peptides, nucleic acidmolecules, carbohydrates, sugars, lipids, lipoproteins, small molecules,natural and synthetic organic and inorganic materials, syntheticpolymers, and the like.

As used herein, a “receptor” refers to a molecular structure within acell or on the surface characterized by (1) selective binding of aspecific substance and (2) a specific physiologic effect thataccompanies the binding, e.g., membrane receptors for peptide hormones,neurotransmitters, antigens, complement fragments, and immunoglobulinsand nuclear receptors for steroid hormones and include natural andsynthetic biomolecules, such as proteins, polypeptides, peptides,nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins,small molecules, natural and synthetic organic and inorganic materials,synthetic polymers, and the like.

As used herein, “specifically binds” refers to the character of areceptor which recognizes and interacts with a ligand but does notsubstantially recognize and interact with other molecules in a sampleunder given conditions.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids”, whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

An “isolated” nucleic acid molecule or polypeptide refers to a nucleicacid molecule or polypeptide that is in an environment that is differentfrom its native environment in which the nucleic acid molecule orpolypeptide naturally occurs. Isolated nucleic acid molecules orpolypeptides includes those having nucleotides or amino acids flankingat least one end that is not native to the given nucleic acid moleculeor polypeptide. For example, a promoter P for a protein X is inserted atthe 5′ end of a protein Y which does not natively have P at its 5′ end.Protein Y is thus considered to be “isolated”.

As provided herein, the expression vectors and libraries of the presentinvention incorporate (1) the use of a regulatable expression vectorthat allows on-off control of carrier polypeptide production, (2)efficient restriction sites immediately adjacent to the randomized siteto facilitate high-efficiency cloning, (3) random polypeptides insertedinto non-conserved sites of carrier polypeptide extracellular loops thatefficiently presents a passenger polypeptide to an given ligand, (4)time and temperature-controlled induction periods to obtain optimaldisplay level that result in higher quality results, (5) the use of abacterial strain having a high plasmid transformation efficiency fortransformation, the use of optimized library construction protocols toconstruct the largest libraries, (6) the use of multiple-plasmidtransformation to yield a larger number of unique passenger polypeptidesfor a given number of host cells, (7) the use of cell concentration toenable complete processing of larger numbers of sequences (10¹¹), (8)the use of gene encoding the carrier polypeptide deficient in one ormore amino acids, or (9) a combination thereof.

The present invention may be broadly applied to methods to isolate,improve or otherwise alter, peptide and polypeptide sequences thatperform useful or desired functions including binding, catalysis,assembly, transport, and the like. For example, the expression vectorsof the present invention may be used to isolate peptide moleculartransformation catalysts, develop whole-cell reagents, discover peptidesthat promote self assembly, discover in vivo targeting peptides for drugand gene delivery, discover and improve peptides binding to materialssurfaces, e.g., semiconductors, mapping proteins such as proteincontacts, and biomolecular networks, identifying enzymesubstrates/inhibitors, identifying receptor agonists/antagonists,isolating inhibitors of bacterial or viral pathogenesis, discoveringpeptides that mediate endocytosis and cellular entry, mapping antibodyand protein epitopes including multiplex mapping, identifying peptidemimics of non-peptide ligands, and isolating metal binding peptides,e.g., for bioremediation, nano-wire synthesis, according to methodsknown in the art. See Georgiou, G., et al. (1997) Nat. Biotechnol.15(1):29-34; Pasqualini, R. and E. Ruoslahti (1996) Nature380(6572):364-366; Whaley, S. R., et al. (2000) Nature405(6787):665-668; Fields, S, and R. Sternglanz (1994) Trends inGenetics 10(8):286-292; Kim, W. C., et al. (2000) J. Biomol. Screen.5(6):435-440; Yang, W. P., et al. (1995) J. Mol. Biol. 254(3): 392-403;Poul, M. A., et al. (2000) J. Mol. Biol. 301(5):1149-1161; James, L. C.,et al. (2003) Science 299(5611):1362-1367; Feldhaus, M. J., et al.(2003) Nat. Biotechnol. 21(2):163-170; Kjaergaard, K., et al. (2001)Appl. Environ. Microbiol. 67(12):5467-5473, and Shusta, E. V., et al.(1999) Curr. Opin. Biotechnol. 10(2):117-122, which are hereinincorporated by reference.

As provided herein, the expression vectors of the present invention maybe used to elucidate consensus sequences while maintaining diversity inselected populations according to methods known in the art. See Smith,G. P. and A. M. Fernandez (2004) Biotechniques 36(4):610-614, 616, 618;and Lowman, H. B. (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401-424,which are herein incorporated by reference.

The present invention provides library sizes (about 5×10¹⁰) that areabout 10-fold larger than typical phage display peptide libraries, withsome exceptions. See Deshayes, K., et al. (2002) Chem. Biol.9(4):495-505; and Fisch, I., et al. (1996) PNAS USA 93(15):7761-7766,which are herein incorporated by reference.

As provided herein, the relatively long 15-mer passenger polypeptidesmay increase the frequency at which high affinity binders occur relativeto the prior art which enables longer consensus motifs and secondarystructures to be determined. See Nakamura, G. R., et al. (2002) PNAS USA99(3):1303-1308, which is herein incorporated by reference.

The expression vectors of the present invention used in conjunction withFACS provides fine discrimination of clonal affinity, and quantitativeseparations that take advantage of this sensitivity. See Van Antwerp, J.J. and K. D. Wittrup (2000) Biotechnol. Prog. 16(1):31-37; andDaugherty, P. S., et al. (1998) Protein Eng. 11(9):825-832, which areherein incorporated by reference. Specifically, the fine affinitydiscrimination provided by FACS allowed isolation of the best sequencesbinding to streptavidin, CRP, and anti-T7•tag Mab. Further, the displaysystems herein routinely enabled identification of beneficial cysteineplacements to form putative disulfide constrained loops conferring highbinding affinity without explicit library design, which alleviates theneed to construct and screen twenty or more different libraries, andremoves critical assumptions that have limited the affinities ofisolated ligands in earlier studies. See Giebel, L. B., et al. (1995)Biochemistry 34(47): 15430-15435; Deshayes, K., et al. (2002) Chem.Biol. 9(4):495-505; and Nakamura, G. R., et al. (2002) PNAS USA99(3):1303-1308, which are herein incorporated by reference.

For example, bacterial display selections for binding to streptavidinyielded a strong preference for CxxVC ligands in all rounds ofselection. Yet, only a single report has described the generation andscreening of a CxxxC type library using phage display technology.Putative disulfide loops were present in peptides binding to all five ofthe targets tested despite about a 1000-fold reduced probability ofoccurring randomly. See FIG. 1. FIG. 1 shows isolated sequencespossessing putative disulfide constrained loops. While a strongconsensus sequence of NxRGF was present in clones from the selection forCRP binding, FACS screening of the enriched pool resulted in theisolation of a peptide (CRP-1) having the identical consensus, butflanked by two cysteines (EWA-CNDRGFNC-QLQR (SEQ ID NO:1)). Though ahandful of previous studies have reported the identification of peptideswith non-designed disulfide bridges, linear libraries most often resultin non-cyclic peptides. See Sahu, A., et al. (1996) J. Immunol.157(2):884-891; and Lu, D., et al. (2003) J. Biol. Chem. 12:12, whichare herein incorporated by reference.

The fact that cyclic peptides were found among the highest affinityclones for all of the ligands tested herein further underscores theimportance of ligand rigidity in high affinity binding. Thus, thepresent invention provides construction of a single library ofsufficient size and quality enables routine isolation of high affinitycyclic peptides. For the construction of intrinsically fluorescentlibraries, a ribosomal binding site (RBS) known in the art may beintroduced downstream of the carrier protein, e.g., OmpX, OmpA, and thelike, followed by a suitable fluorescent protein, e.g., alajGFP. SeeBessette, P. H. and P. S. Daugherty (2004) Biotechnology Progress 20(1), which is herein incorporated by reference. The resulting bacteria,when expression is induced by the addition of 0.2% arabinose, are bothintrinsically green and display passenger polypeptides as N or Cterminal fusion proteins. See FIG. 7. Alternatively, the order may bereversed such that the fluorescent protein is expressed first, followedby the RBS and the permuted OMP sequence.

Sequences with about 10 to about 100 fold higher affinity may beobtained by randomization of non-consensus residues and kinetic FACSselection (using biotin as a competitor). Streptavidin binding peptidesmay be used as genetically encoded biotin mimics to eliminate the needfor chemical labeling of proteins with biotin. Thus, a streptavidinbinding peptide selected and affinity matured using this process couldbe fused, using recombinant methods known in the art, to either the C orN terminus of at least one given nucleic acid molecule. Expression ofthe nucleic acid molecule would produce a polypeptide having a C orN-terminal peptide tag capable of binding to the commonly used affinityreagent, streptavidin, which may be eluted from the reagent by thesimple addition of biotin.

The polypeptide display systems of the present invention allow thecreation of renewable whole cell binding reagents in non-specializedlaboratories since this method is technically accessible and librariesare reusable. This approach has already proven useful for selectingcell-specific binding peptides, and for performing diagnostic assaysusing flow cytometry and fluorescence microscopy (unpublished data).Furthermore, the surface displayed polypeptides can be used for parallelor multiplex ligand isolation, and clones can be processed withefficient single-cell deposition units present on many cell sorters. SeeFeldhaus, M. J., et al. (2003) Nat. Biotechnol. 21(2):163-170, which isherein incorporated by reference. Consequently, the expression vectorsof the present invention may be used in proteomic applications includingproteome-wide ligand screens for protein-detecting array development.See Kodadek, T. (2001) Chem. Biol. 8(2):105-115, which is hereinincorporated by reference.

A. OmpA Loop 1 Expression Vector

While a purpose of “cell surface display” systems is to presentpolypeptides on living cells to extracellular targets of any size andmolecular composition, LppOmpA′, periplasmic display (PECS), andanchored periplasmic expression (APEx) systems, in the prior art do notenable this objective. See Stathopoulos, C., et al. (1996) Appl.Microbiol. & Biotech. 45(1-2): 112-119; Lang, H. (2000) Int. J. Med.Microbiol. 290(7):579-585; Lang, H., et al. (2000) Eur. J. Biochem.267(1):163-170; Lang, H., et al. (2000) Adv. Exp. Med. Biol.485:133-136; and Chen, G., et al. (2001) Nat. Biotechnol. 19(6):537-542;Harvey, B. et al. (2004). PNAS. 101(25) 9193-9198, which are hereinincorporated by reference. Surface display with LppOmpA′ refers to theuse of a genetic fusion to localize a polypeptide to the outer membraneof E. coli, though not necessarily in a manner that enables binding toarbitrary extracellular targets. Periplasmic display and outer membranelocalization with LppOmpA′ do not present the displayed protein in amanner compatible with binding to extracellular macromolecules except inrare examples. See Francisco, J. A., et al. (1992) PNAS USA89(7):2713-2717; Stathopoulos, C., et al. (1996) Appl. Microbiol. &Biotech. 45(1-2):112-119; Francisco, J. A., et al. (1993) PNAS USA90(22):10444-10448; Francisco, J. A., et al. (1993) Bio/Technology11(4):491-495; Francisco, J. A. and G. Georgiou (1994) Annals NY Acad.Sci. 745:372-382; and Georgiou, G., et al. (1993) Trends InBiotechnology 11(1):6-10, which are herein incorporated by reference.

In both of these prior art systems, the displayed protein can interactonly with molecules that penetrate the outer membrane, e.g., small andtypically hydrophobic molecules, and not with any known protein ormacromolecule. This precludes application of the prior art displaysystems in a wide range of commercially and medically importantapplications, e.g., protein diagnostics, sensing, and proteomics,cellular array construction, cellular targeting, materials science andmaterials surface functionalization with whole cells, and the like.Surface localization via a membrane targeting sequence, e.g., the signalsequence and amino acids 1-9 of Lpp, results in membrane disruption andconsequently reduced cell growth rates and viability. Application ofperiplasmic expression (PECS) or anchored periplasmic expression (APex)for protein library screening would require that the cell membrane isremoved prior to addition of the target ligand causing cell death.Polypeptide encoding genes on plasmids contained within the cells mustthen be isolated, PCR amplified to recover genes encoding thecorresponding polypeptide, and sub-cloned into an expression vector forlibrary enrichment and repeat screening or selection. Consequently, thisapproach is much slower, most costly, and less effective than thepresent system.

In contrast, the OmpA loop 1 expression vector, MC1061/pBAD33L1, of thepresent invention exemplified herein presents polypeptides at about theoutermost point of the first loop of OmpA which increases distance fromthe lipopolysaccharide surface of E. coli, thereby reducingelectrostatic repulsion and steric hindrance between the target element,e.g., protein, and the displayed polypeptide and provides efficientrecognition of macromolecules, inorganic surfaces, and cell surfaces.MC1061/pBAD33L1 differs from the previously reported vector utilizingLMG19/pB30D in several important aspects that change the function ofthis system. See Daugherty, P. S., et al. (1998) Protein Eng.11(9):825-832; Daugherty, P. S., et al. (1999) Protein Eng.12(7):613-621; Daugherty, P. S., et al. (2000) J. Immunol. Methods243(1-2):211-227; and Daugherty, P. S., et al. (2000) PNAS USA97(5):2029-2034, which are herein incorporated by reference.

The present expression vectors and libraries utilize an extracellularloop of monomeric outer membrane protein (e.g., OmpA & OmpX), which isaccessible to arbitrary compositions of matter, capable of beingproduced at high levels in the outer membrane to enable to best andpreferred modes of selection and screening. Polypeptide encoding DNAsequences are inserted genetically within the Omp gene corresponding tothe outermost point of loop exposure to the extracellular environment.In some preferred embodiments, this is in the first extracellular loopof OmpA between LIGQ-X-(X)_(n)-NGPT (SEQ ID NO:2) wherein X is an aminoacid and n is any positive integer, as shown in FIG. 2. In contrast,LppOmpA46-159 (LppOmpA′), utilizes a fusion to the newly generatedC-terminus resulting from truncation of the OmpA protein at amino acid159. The benefit of the insertional fusion of the present invention isthat it preserves the stability of the overall topological structure ofouter membrane protein. Structure is preserved in the construct of thepresent invention, since adjacent beta-strands maintain molecularinteractions that confer stability to the Omp barrel structure. Also,the insertion sites in OmpA are designed by consideration ofnon-conserved sequences in loops indicating tolerance to substitutionand thus insertion.

To enable construction of a highly diverse polypeptide display library,two mobile loops of OmpA were compared for their ability to display a15-amino acid epitope. See FIG. 3. E. coli OmpA was chosen as a displayscaffold since (1) it is monomeric and can be produced at high levels inthe outer membrane under certain conditions; (2) the structuresdetermined using x-ray crystallography and NMR indicate the presence offlexible extracellular loops, and (3) it has been shown to accept loopinsertions. See Pautsch, A. and G. E. Schulz (2000) J. Mol. Biol.298(2):273-8229; Arora, A., et al. (2001) Nat. Struct. Biol. 8(4):334-830; Freudl, R. (1989) Gene 82(2):229-3631; Mejare, M., (1998)Protein Eng. 11(6):489-9432; Etz, H., et al. (2001) J. Bacteriol.183(23):6924-6935, which are herein incorporated by reference.

The site of insertion of the display systems of the present inventiondoes not hinder the export of a diverse range of protein and peptidesequences yet retain structural stability. Since loops 1 and 4 arethought to be relatively flexible, it was reasoned that they would beless likely to adversely impact structural stability. Consequently, a15-mer insertional fusion containing the 11 amino acid epitope of T7gene 10 (T7•tag) (MASMTGGQQMG) (SEQ ID NO:3) was made in each loop atpositions maximally distant from the cell surface within a sequenceregion poorly-conserved among OmpA homologs. Labeling of whole cellswith a biotinylated anti-T7•tag monoclonal antibody (mAb) followed bysecondary labeling with streptavidin-phycoerythrin (SAPE) demonstratedthat both loops were capable of displaying the T7 epitope with differentefficiencies.

Insertions into loop 4 after residue 150 resulted in relatively lowlevel display, since fluorescence signals were only about 2-fold greaterthan background cellular autofluorescence. On the other hand, loop 1epitope insertions after residue 26 (FIG. 3) resulted in efficientT7•tag display, with cells exhibiting 300-fold increased fluorescenceabove background control cells as measured by flow cytometry. Thoughthese experiments were carried out in strain MC 1061, which is ompA⁺,the over-expression of the engineered OmpA was easily detectable and didnot improve in an otherwise isogenic ompA⁻ host.

As provided in Example 1, one of the important features of the OmpA loop1 expression vector of the present invention is that a given polypeptideis located in the first extracellular loop of OmpA which is important as(1) the stability of the overall topological structure of OmpA ispreserved since the adjacent β-strands are required to maintain theoverall stability of the OmpA barrel structure, (2) the polypeptides areproperly expressed on the outer surface of the host cell membrane, and(3) large polypeptides may be expressed. Expression and display of apolypeptide using the OmpA loop 1 expression vector exhibits reduced(wild-type-like) membrane permeability to toxic agents which improvesviability and growth rates.

In the OmpA loop 1 expression vector exemplified herein, the DNAsequence encoding the polypeptide to be expressed is inserted betweenthe native OmpA sequences that encode amino acid residues N25 and N27(with numbering with respect to the mature protein); however, it shouldbe noted that OmpA loop 1 expression vectors having other insertionsites within loop 1 are contemplated and may be constructed according tothe present invention. See Table 1.

TABLE 1 Loop 1 OmpA homologs from other species suitable for polypeptidedisplay (preferred insertion locations in bold) Organism FirstExtracellular Loop Sequence Esherichia coliAKLGWSQYHDTGFINNN-----GPTHENQLGAGA (SEQ ID NO: 4) Esherichia coliAKLGWSQYHDTGLINNN-----GPTHENQLGAGA (SEQ ID NO: 5) Shigella sonneiAKLGWSQYHDTGFINNN-----GPTHENQLGAGA (SEQ ID NO: 6) ShigellaAKLGWSQYHDTGFIDNN-----GPTHENQLGAGA dysenteriae (SEQ ID NO: 7) ShigellaAKLGWSQYHDTGFIPNN-----GPTHENQLGAGA flexneri (SEQ ID NO: 8) SalmonellaAKLGWSQYHDTGFIHND-----GPTHENQLGAGA typhimurium (SEQ ID NO: 9) SalmonellaAKLGWSQYHDTGFIHND-----GPTHENQLGAGA enterica (SEQ ID NO: 10) EnterohacterAKLGWSQFHDTGWYNSNLNNN-GPTHESQLGAGA aerogenes (SEQ ID NO: 11) YersiniaPestis AKLGWSQYQDTGSIINND----GPTHKDQLGAGA (SEQ ID NO: 12) KlebsiellaAKLGWSQYHDTGFYGNGFQNNNGPTRNDQLGAGA pneumoniae (SEQ ID NO: 13)

In preferred embodiments of the present invention, the OmpA loop 1expression vector displays polypeptides on about the outermost point ofthe first loop of OmpA which increases distance from thelipopolysaccharide surface of the host cell and consequently reducedelectrostatic repulsion and steric hindrance between the target element,e.g. protein, and the displayed polypeptide. In some embodiments of thepresent invention, the nucleic acid sequence of the expression vectorwas changed to N25Q (to introduce SfiI with a conservative amino acidreplacement) and the nucleic acid sequence for N26 was deleted.

Some preferred sites insertion of a given polypeptide may be determinedusing methods known in the art including analysis of crystal structure,sequence, NMR structure, and then tested using peptide epitopes known tobe recognized using common anti-peptide antibodies, e.g., the T7antibody, anti-c-myc, anti-HA, anti-FLAG. An example of an ideal sitefor gene insertion is the first extracellular loop of Escherichia coliOmpA between residues Asn-Asn-Asn (SEQ ID NO:14).

Alternative insertion sites include loop 1 OmpA homologs, which may beidentified by multiple sequence alignment to identified non-conservedregions and is preferably chosen such that the displayed protein islocated more than about 1 nM from the outer membrane of the cell. Seee.g. Table 1.

Other features of the expression vector of the present invention include(1) the use of the OmpA signal sequence, (2) two SfiI restriction siteswith one located in OmpA loop 1 immediately adjacent to the insertionsite and a second assymetric SfiI located at an arbitrary distance, butopposite of the insertion site relative to the first SfiI, (3) a singleresistance gene for a bacteriocidal antibiotic such as chloramphenicolacetyltransferase, (4) a low copy origin of replication such as p15A forlow level expression, and (5) a regulatable promoter, such as araBADpromoter, for controlled transcription.

1. OmpA Signal Sequence

pBAD33L1 utilizes the OmpA signal sequence, rather than the Lpp leadersequence employed in LppOmpA, thus providing optimal secretion throughthe inner membrane.

2. Restriction Enzyme (SfiI) Cleavage Sites

As provided herein, the vector design incorporates two SfiI sitesdirectly into the Omp reading frame and provides a minimized size whichpermits libraries of a preferred size, about 10⁸ to about 10¹², to beefficiently constructed and used. Specifically, pBAD33L1 contains SfiIrestriction sites engineered directly into OmpA loop 1 and 4, therebyenabling high efficiency insertion of cloned genes and large libraryconstruction.

The SfiI restriction sites allow the introduction of a nucleic acidmolecule which can be digested by a particular enzyme but generatesoverhangs which cannot react with incorrect DNA substrates, e.g.,GGCCXXXXXGGCC (SEQ ID NO:15), which is recognized by the restrictionendonuclease, SfiI, about 1 to about 50 bp upstream of the site wherethe display molecules will be introduced (the insertion site), and thesite GGCCXXXXXGGCC (SEQ ID NO:15) at a distance of about 300 to about1500 by downstream of the insertion site. This method permits use ofsynthetic randomized oligonucleotides that incorporate the same SfiIsequence to be used in a polymerase chain reaction to create sufficientnumbers of random DNA fragments.

3. Bacteriocidal Resistance Gene

pBAD33L1 contains only a single resistance gene encoding chloramphenicolacetyltransferase, rather the both cat and beta lactamase. The plasmid,pBAD33L1, is therefore smaller, thereby providing greater transformationefficiency than pB30D. Importantly, owing to size and absence ofbeta-lactamase expression pBAD33L1 imposes a smaller burden upon cellgrowth than previous vectors, thereby improving library screening.Further the ability to use a bacteriocidal antibiotic for selection ispreferred in order to prevent plasmid loss and the outgrowth ofbacterial cells commonly resistant to the antibiotic.

4. Low Copy Origin of Replication

The use of a low copy plasmid utilizing the p15A origin of replicationenabled expression without a significant reduction of cell viability.See FIG. 4. In contrast, an analogous display vector having a pMB1origin provided high level expression but resulted in rapid arrest ofcell growth shortly after induction (data not shown). In someembodiments, expression of the displayed protein does not hinder cellgrowth in order to prevent clonal competition that reduces librarydiversity and interferes with selection. As an alternative to using alow copy plasmid, a higher copy plasmid, e.g., plasmid containing thepMB1 origin of replication, could be used in combination with a promoterhaving reduced transcriptional activity.

5. Regulatable Promoter

The expression vectors of the present invention incorporate a tightlycontrolled promoter for regulated transcription. As exemplified herein,the promoter used is from the arabinose araBAD operon of E. coli. SeeGuzman, L., et al. (1995) J. Bacteriol. 177(14):4121-4130; Johnson, C.M. and R. F. Schleif (1995) J. Bacteriol. 177(12):3438-3442; Khlebnikov,A., et al. (2000) J. Bacteriol. 182(24):7029-7034; and Lutz, R. and H.Bujard (1997) Nucleic Acids Res. 25(6):1203-1210, which are hereinincorporated by reference. Protein production is initiated by additionof the sugar L-arabinose, and stopped by the removal of arabinose andaddition of glucose. Regulation prevents unwanted changes in therepresentation frequency of the rare desired target cells during growthbefore and after the selection or screening step.

The use of a tightly regulatable promoter prevents loss of mildly toxicsequences during growth, maintain full library diversity, and improvesingle round enrichment efficiency. See Daugherty, P. S., et al. (1999)Protein Eng. 12(7):613-621, which is herein incorporated by reference.Use of the arabinose inducible promoter from the araBAD operon enabledtight repression in the absence of arabinose during library propagationand reproducible induction of surface display of peptide insertionsunder saturating inducer conditions. High level display with minimalcell death or growth inhibition (data not shown) was obtained about 1 toabout 4 hours after induction. In subsequent experiments, an inductionperiod of about 2 hours was typically used before selection or screeningto minimize potential toxicity.

Any promoter could be used according to the present invention that (1)provides tight repression of expression during library propagationbefore and after screening, and (2) provides adequate levels ofexpression to enable binding magnetic particles and or be detected usingflow cytometry instrumentation. In alternative embodiments, amodulatable promoter may be used, which enables “rheostat” control ofexpression over a range of potentially desirable expression levels.Examples of such promoters include, the araBAD system, withco-expression of a constitutive arabinose transporter protein. SeeKhlebnikov, A., et al. (2000) J. Bacteriol. 182(24):7029-7034, which isherein incorporated by reference.

As disclosed in Example 1, for library construction of the OmpA loop 1expression vector, inserts were chosen to have a length of about 15codons while allowing all possible amino acids (using NNS degeneratecodons) at each position. In addition to increasing the physicaldistance from the cell surface, longer length insert libraries, e.g.,15-mer, offer the advantage of providing more copies of short sequenceswhile allowing for longer binding motifs to emerge. The resultinglibrary of about 5×10¹⁰ independent transformants provides a sparsesampling of the sequence space available to a 15-mer (0.0000002%), butis expected to contain all possible 7-mer sequences (greater than about99% confidence).

In some embodiments of the present invention, a DNA library isconstructed containing preferably greater than about 10⁸ sequences, andpreferably more than about 10¹⁰ unique sequence members, using methodsknown in the art. This library size is preferred since library size hasbeen shown to correlate with the quality (affinity and specificity) ofthe selected sequences. See Griffiths, A. D. and D. S. Tawfik (2000)Curr. Opin. Biotechnol. 11(4):338-53, which is herein incorporated byreference.

In some embodiments, a polypeptide library may prepared by introductionand expression of nucleic acid sequences which encode polypeptideshaving about 1 to about 1000, preferably about 2 to about 30 amino acidsin length. As provided herein, the present invention uses high DNAconcentrations of more than about 0.1 μg per μl during transformationwhich resulted in one or more independent plasmid molecules in each hostcell. This multiple-plasmid transformation step, yields a larger numberof unique peptides in the same volume of liquid, providing the overallresults better than prior art methods which provide only one moleculeper cell. In some embodiments, a mixture of a plurality of differentexpression vectors and/or plasmids may be employed to providecooperative binding two different displayed peptides on the samesurface, to present a protein having multiple subunits, and the like.

A desired number of polypeptides may be displayed on the surface fordifferent purposes. As exemplified herein, the method of the presentinvention utilizes an induction period of about 10 minutes to 6 hours tocontrol total expression levels of the display polypeptide and the modeof the subsequent screen or selection such that the level of expressionhas no measurable effect upon the cell growth rate. See FIG. 4. In someembodiments, shorter time periods may be used to reduce avidity effectsin order to allow selection of high affinity monovalent interactions. Asprovided herein, the ability to control display speeds the process andyields higher quality results, e.g., sequences that bind to a targetwith higher affinity.

In some embodiments, a cell concentration by a factor of about 10 may beused to enable complete processing of the entire pool of diversity in avolume of about 10 to about 100 ml. The library may be expanded bypropagation by a factor of more than about 100-fold under conditionswhich prevent synthesis of the library elements, for example, withglucose to repress the araBAD or lac promoters, and aliquots of thelibrary may be prepared to represent a number of clones which is morethan about three fold greater than the total number of library members.See FIG. 5.

For library selection, a subset of the total library, either randomlydivided, or chosen for specific properties could be used as a startingpoint for screening. Either MACS or FACS methods known in the art may beused, in place of sequence application of MACS and then FACS. As analternative to FACS, methods known in the art that enable physicalretention of desired clones and dilution or removal of undesired clonesmay be used. For example, the library may be grown in a chemostatproviding continuous growth, diluting out only those cells that do notbind to a capture agent retained in the vessel. Alternatively, hosts maybe cultured with medium having ingredients that promote growth ofdesired clones.

Instead of using random synthetic peptides to provide genetic diversity,fragment genomic DNA of varying lengths, cDNA of varying lengths,shuffled DNAs, and consensus generated sequences may be employed inaccordance with the present invention.

Non-natural amino acids having functionality not represented amongnatural amino acids, e.g., metal binding, photoactivity, chemicalfunctionality, and the like, may be displayed on the surface using asuitable host. In this case, the library or an equivalent library may betransformed into strains engineered to produced non-natural amino acids.See Kiick, K. L. et al. (2001) FEBS Lett. 502(1-2):25-30; Kiick, K. L.,et al. (2002) PNAS USA 99(1):19-24; Kirshenbaum, K., et al. (2002)Chembiochem. 3(2-3):235-237; and Sharma, N., et al. (2000) FEBS Lett.467(1):37-40, which are herein incorporated by reference. Peptidesincorporating non-natural amino acids are isolated by selection orscreening for functions which require inclusion of the non-naturalmonomers into the displayed polypeptide.

Displayed polypeptides may be made to include post-translationmodifications, including glycosylation, phosphorylation, hydroxylation,amidation, and the like, by introduction of a gene or set of genesperforming the desired modifications into the strain used for screeningand selection, e.g., MC1061 or comparable host strain. Genes performingsuch post-translational modifications may be isolated from cDNA orgenomic libraries by cotransformation with the library and screening forthe desired function using FACS or another suitable method. For example,post-translational glycosylation activities (enzymes) can be foundco-transforming.

The polypeptides displayed by a carrier protein preferably possess alength that preserves the folding and export of the carrier protein,such as OmpA, OmpX, or the like, while presenting significant sequenceand structural diversity. In some embodiments, the carrier protein, suchas an outer membrane protein (Omp), may be modified by rational redesignor directed evolution methods known in the art to increase levels ofdisplay or improve polypeptide presentation. For example, the carrierprotein may be optimized by random point or cassette mutagenesis andscreening for improved presentation. Sequences not required for display,such as the C-terminal domain of OmpA, may be removed from the displaycarrier protein in order to minimize metabolic burden and improve totaldisplay levels.

In some embodiments, an alternative Omp, such as OmpX, OmpF, LamB, OmpC,OmpT, OmpS, FhuA, FepA, FecA, PhoA, and TolC, may be used as the carrierprotein. Epitope insertion assays known in the art, and here exemplifiedby the insertion of the T7tag peptide into OmpA, OmpX, and CPXpolypeptides, may be used to identify suitable passenger insertion sitesconferring display at the surface. Growth assays known in the art, maybe used to identify insertion sites which do not alter growth rates orviability as a result of display. Sec FIG. 4.

Multimeric membrane proteins could be used, either in native form forpolyvalent display, e.g., three peptide on trimeric OmpF, or could beengineered to be monomeric, thereby mimicking OmpA, OmpX, or OmpT. SeeFIG. 2 and FIG. 6. However, in preferred embodiments, display is via amonomeric protein, e.g., OmpA, OmpX, or catalytically inactive mutant ofOmpT, present at the cell surface in excess of 10,000 copies per cell.

In some embodiments, an alternative protein scaffold protein may be usedto present the passenger polypeptide, e.g., random peptide, to bedisplayed. For example the green fluorescent protein or an alpha helixbundle protein, knottin, acylic permutant of a cyclic peptide (e.g.,Kalata-B1) may be used as a spacer and scaffold element, or to providemultiple additive or synergistic functions, e.g., fluorescence &binding, binding, and catalytic transformation, binding and assembly,and the like. See FIG. 7.

As exemplified herein, the present invention utilizes a bacterialstrain, MC1061 which exhibits (1) high plasmid transformation efficiencyof greater than about 5×10⁹ per microgram of DNA, (2) a short doublingtime, i.e., 40 minutes or less, during exponential growth phase, (3)high level display of the given polypeptide, and (4) effectivemaintenance of the expression ON and OFF states. See FIG. 3, FIG. 4, andFIG. 5. In some embodiments, alternative biological entities known inthe art may be used. In preferred embodiments, the biological entity isdeficient in proteolytic machinery in order to prevent proteindegradation. See Meerman, H. J., Nature Biotechnol. 12(11):1107-1110,which is herein incorporated by reference. In some embodiments, abiological entity that makes truncated or otherwise modifiedlipopolysaccharides on its surface may be used to minimize stericeffects upon binding to large biomolecules including proteins, viruses,cells, and the like. In some preferred embodiments, the biologicalentity has a genotype that aids the expression vector in regulating moretightly the production of the polypeptide to be displayed. Thebiological entity may be modified using methods known in the art,including random mutagenesis, DNA shuffling, genome shuffling, geneaddition libraries, and the like.

As provided herein, expression and display of the polypeptide may beaccomplished by induction of protein expression by contacting witharabinose, preferably about 10 to about 60 minutes, and more preferablyabout 10 to about 20 minutes at 25° C. Controlling expression anddisplay minimizes potential avidity effects that can result fromexcessive surface concentration of the displayed peptide. Cells weregrown in LB media overnight or for about 1 to about 3 hours, induced forabout 5 minutes to about 5 hours at about 4 to about 37° C., andpreferably for about 10 to about 20 minutes at about 25° C. Cells werewashed once in phosphate buffered saline (PBS) and resuspended in PBSwith biotin conjugated target protein. The cells were then washed onceto remove unwanted unbound proteins and other debris, and incubated witha fluorescent, biotin binding reagent, preferablystreptavidin-phycoerythrin (Molecular Probes, Eugene, Oreg.), or thelike. Unbound fluorescent reagent was then removed by washing and cellswere analyzed by flow cytometry. Cells displaying the foreign protein orpeptide possess a fluorescence intensity of about 8 to about 200 foldgreater than non-peptide display cells, i.e., cellular autofluorescence,and preferably about 8 to about 20 fold, indicating a moderate level ofdisplay that will not result in avidity effects (about 1000 to about10,000 copies).

As provided herein, use of MC1061/pBAD33L1 allowed the identification ofoptimal disulphide bond placements in selected peptides directly fromlarge random libraries which increases the affinity of the selectedligands, and provides utility for applications requiring stability,e.g., serum stability in vivo. The display systems of the presentinvention allow the use of magnetic selection which provides relativelysimple and fast, e.g., about 2 days, isolation of peptides that bind toa target ligand with high binding affinity.

As provided in Example 1, the ability of the displayed polypeptides tobind given ligands was tested. Five unrelated target proteins werechosen: a monoclonal IgG antibody binding to a known epitope(anti-T7•tag mAb), human serum albumin (HSA), human C-reactive protein(CRP), streptavidin, and HIV-1 GP120. For each of five protein targetstested, magnetic selection enabled enrichment of clones displayingprotein binding peptides from non-binding clones. Abundant streptavidinbinding peptides were first depleted from the library using one round ofmagnetic selection with streptavidin functionalized magnetic particles.The remaining cells were incubated with biotinylated target proteins,and subsequently with biotin-binding magnetic particles to capture cellswith bound target protein. Each cycle of magnetic selection was followedby overnight growth to amplify the selected population.

Flow cytometry was used to monitor the progress of magnetic selectionusing as fluorescent probes either streptavidin-phycoerythrin orfluorescently conjugated anti-biotin antibodies. See FIG. 9. One or tworounds of magnetic selection were sufficient to enrich a populationcontaining a significant fraction of binders for each of the fivetargets tested. In the case of selection for anti-T7•tag mAb binding,one cycle was sufficient to enrich binding peptides to nearly about 50%of the population from an initial frequency of about 1 in 50,000—asingle round enrichment of about 25,000-fold. The initial frequency ofT7•tag mAb binding clones indicated that roughly about 2×10⁵ uniquepeptide sequences were capable of binding when using a targetconcentration of 10 nM.

The frequency of target protein binding peptides within the librarypopulation was found to vary significantly among different targets,suggesting that the library was more “fit” for binding some antigens.The highest frequency of target binding cells was observed with theanti-T7•tag mAb. Similarly, a high initial frequency of positive cellswas observed when using streptavidin and CRP as targets. On the otherhand, a reduced frequency (less than about 1:10⁶) of GP120 bindingclones was observed in the unselected library, possibly reflecting theheavily glycosylated surface of this target. The frequency of targetbinding clones in the library was consistent with the probability ofoccurrence of certain critical motifs involved in molecular recognition.In the anti-T7•tag mAb selection, for example, the initial frequencybinding clones (2:10⁵) is consistent with the expected frequency of theidentified “core” motif MxP(x/-)QQ of about 2:10⁵. Similarly, for theCRP selection, the consensus motif, NxRGF, is expected to occur at afrequency of roughly about 5:10⁵. Thus, cytometric analysis of thelibrary populations prior to screening provided useful statisticalinformation regarding the expected frequency of target protein-bindingpeptides.

Cell sorting instrumentation was applied as a quantitative libraryscreening tool to isolate the highest affinity clones from themagnetically enriched populations (FIG. 3), estimated to represent about10⁵ to about 10⁷ unique sequences. Two fundamentally differentapproaches were applied for quantitative screening, as previouslydescribed, on the basis of either equilibrium binding affinity(Equilibrium Screen) or dissociation rate constants (Kinetic Screen).See Daugherty, P. S., et al. (2000) J. Immunol. Methods243(1-2):211-227; and Boder, E. T. and K. D. Wittrup (1998)Biotechnology Progress 14(1):55-62, which are herein incorporated byreference. In most cases, appropriate antigen concentrations forequilibrium screening were determined by flow cytometric analysis ofabout 10⁶ clones after labeling with a range of different target proteinconcentrations.

For equilibrium screening, cell populations were labeled with limitingconcentrations of the target proteins, and all cells exhibitingfluorescence intensities above background autofluorescence werecollected. See FIG. 8. Thus, the ligand concentration, and not the lowerintensity limit of the sort window, was used to as the criteria foracceptance. In the case of the streptavidin selection, kinetic screeningwas performed using free biotin as a competitor. In the absence ofbiotin, streptavidin binding peptides exhibited substantially slowerdissociation rates, likely due to rebinding effects. The apparentbinding affinities of isolated clones were generally predictable fromthe antigen concentrations used for screening Typically, the apparentdissociation constants were roughly ten-fold higher than the ligandconcentration used for screening. See Table 2.

TABLE 2 Target Conc. Clone (K_(D)) Sequence (nM)^(a) C-Reactive ProteinCRP-1 (1 nM)     EWACNDRGFNCQLQR SEQ ID NO: 16 0.1 CRP-2 (3 nM)    FPIYNQRGFITLASP SEQ ID NO: 17 0.1 CRP-3     HMRWNTRGFLYPAMS SEQ IDNO: 18  1.0^(b) CRP-4     RYIMNHRGFYIFVPR SEQ ID NO: 19  1.0^(b) CRP-5    VRTWNDRGFQQSVDR SEQ ID NO: 20  1.0^(b) CRP-6 (8 nM)     MIFNSRGFLSLMSSG SEQ ID NO: 21 10.0  CRP-7       LMNWRGFMVPRESPK SEQID NO: 22 10.0  CRP-8    WTKLKNSRGFELQLD SEQ ID NO: 23 10.0  CRP-9    PYLNARGFSVTREQI SEQ ID NO: 24 10.0  Consensus       IXNXRGF SEQ IDNO: 25 CRP-10 (5 nM)   YPPRFQYYRFYYRGP SEQ ID NO: 26 0.1 CRP-11   TDFLSYYRVYRTPLQ SEQ ID NO: 27  1.0^(b) CRP-12    TFMPSYYRSWGPPPT SEQID NO: 28  1.0^(b) CRP-13     TTCKYYLSCRWRKDL SEQ ID NO: 29 10.0 Consensus        SYYRSY SEQ ID NO: 30 Streptavidin SA-1 (10 nM)   RLEICQNVCYYLGTL SEQ ID NO: 31  6.0^(c) SA-2 (8 nM)   ICSYVMYTTCFLRVYSEQ ID NO: 32  6.0^(d) SA-3 (4 nM)    TVLICMNICWTGETQ SEQ ID NO: 33 6.0^(d) SA-4    VTSLCMNVCYSLTTY SEQ ID NO: 34  6.0^(d) SA-5    YWVCMNVCMYYTARQ SEQ ID NO: 35  6.0^(d) SA-6   LPVWCVMHVCLTSSR SEQ IDNO: 36  6.0^(d) SA-7    NEWYCQNVCERMPHS SEQ ID NO: 37 6.0 SA-8   IMMECFYVCTIANTQ SEQ ID NO: 38 6.0 SA-9    TKVQCTMVCYGMSTT SEQ ID NO:39 6.0 SA-10    SITICWYTCMVQKTA SEQ ID NO: 40 6.0 SA-11   ADTICWYVCTISVHA SEQ ID NO: 41 6.0 Consensus       ICMNVC SEQ ID NO:42 Serum Albumin HSA-1    NPFCSWYRWRNWCTK SEQ ID NO: 43 100.0   HSA-2  RHLYC-WT-WR-WCHFKD SEQ ID NO: 44 100.0         CXWXXWRXW SEQ ID NO: 45HSA-3    SYISTWLNFLFCGQS SEQ ID NO: 46 100.0   HSA-4    NNYSAWLRCLLRAYSSEQ ID NO: 47 100.0   Consensus       SXWLXXLXXXXS SEQ ID NO: 48 HIV-1gp120 GP120-1   GDTWVWYCWYWTRSI SEQ ID NO: 49 15.0  GP120-2     WVCTWNYWTRVTWCL SEQ ID NO: 50 15.0  Consensus      WVXXXXYWTR SEQID NO: 51 GP120-3        PWCWMWTKGRWYYVA SEQ ID NO: 52 0.6 GP120-4     QIQWCWVNHRWSPVV SEQ ID NO: 53 0.6 GP120-5   WVAGYWWCWSVMYRS SEQ IDNO: 54 15.0  GP120-6      TWTWCWRNYIWQLST SEQ ID NO: 55 15.0  GP120-7QEWRQLTRWCWVQIK SEQ ID NO: 56 15.0  GP120-8  QTATVSYWCYWWWKV SEQ ID NO:57 15.0  Consensus         WCWXXXK SEQ ID NO: 55 ^(a)The concentrationused for the final selection. ^(b)Dissociation in presence of 100 nMunbiotinylated CRP for 20 minutes. ^(c)Dissociation in presence of 1 μMbiotin for 2.5 hours. ^(d)Dissociation in presence of 1 μM biotin for 6minutes.

Table 2 shows peptide sequences of isolated clones binding tostreptavidin, CRP, HSA, and GP120. Sequences were aligned using theClustal W algorithm, and consensus residues are shown below each group.For selected clones, the apparent whole cell K_(D) as measured by flowcytometry is indicated.

Consensus sequences were readily apparent for each of the targetproteins after two to three rounds of magnetic selection and one or tworounds of FACS. See Table 2, FIG. 9. The strongest consensus sequencefor anti-T7•tag mAb binding in a single clone was lengthened to sevenresidues SMGPQQM (SEQ ID NO:59), despite the low frequency of suchclones in the library, i.e., about 1:10¹⁰. One anti-T7•tag mAb binder,(FIG. 9) possessed seven identities and one similarity with thewild-type T7•tag sequence. Considering codon usage, such a clone wouldbe expected to occur at a frequency of less than about one in 10¹⁰.Consensus sequences for HSA and for HIV-1 GP120 binding included severalhydrophobic residues, and a high frequency of clones with one or twocysteine residues. In some cases, FACS resulted in enrichment andisolation of putatively cyclic peptides incorporating the consensussequence. For example, the highest affinity CRP binding clone (CRP-1,Table 2) from stringent FACS screening possessed the consensus NxRGFflanked by cysteines—CNDRGFNC (SEQ ID NO:60). Residues outside of thecyclic constrained consensus also contributed to improved function sincetwo streptavidin binding clones with identical disulfide loops (CQNVC(SEQ ID NO:61)) possessed dissociation rate constants differing byfour-fold. See FIG. 10B. The overall length of the visible consensussequences spanned as many as about ten or about eleven residues foranti-T7•tag mAb (SMGPQQMXAW (SEQ ID NO:62) or SMGPQQMAW (SEQ ID NO:63))or CRP (IXNXRGFXXXV (SEQ ID NO:64)), suggesting that libraries withshorter inserts would not have yielded peptides with comparableaffinities, or provided equivalent epitope mapping information.

The apparent binding affinities of a subset of the selected peptideswere determined using flow cytometric analysis. This method has beenshown to enable reliable estimation of both K_(D) and k_(diss) values.See Daugherty, P. S., et al. (1998) Protein Eng. 11(9):825-832, which isherein incorporated by reference. And importantly, the relative affinityranking of selected clones obtained using flow cytometry has been shownto be equivalent to that determined using Surface Plasmon Resonance. SeeDaugherty, P. S., et al. (1998) Protein Eng. 11(9):825-832; andFeldhaus, M. J., et al. (2003) Nat. Biotechnol. 21(2):163-170, which areherein incorporated by reference. Apparent equilibrium dissociationconstants (FIG. 10A) were typically in the low nanomolar range(K_(D)=1-10 nM) (Table A), as determined using fluorescently conjugatedCRP and SA. Similarly, the best GP120 binding clones exhibited highfluorescence after incubation with 10 nM GP120, indicating that theK_(D) is less than about 10 nM (data not shown). Apparent dissociationrate constants (k_(diss)) were determined for streptavidin, using about1 to about 2 μM biotin as a competitor to prevent re-binding.

Rate constants were found to range from about 0.01 s⁻¹ after two cyclesof MACS and one cycle of FACS (clones SA-7 to SA-11) to about 0.001 s⁻¹after an additional round of screening (clones SA-1 to SA-6). Althoughthe potential avidity effects for surface displayed peptides binding tomultimeric target proteins were not ruled out, the dissociation kineticsshow excellent agreement with a single exponential decay (FIG. 10B),suggesting about a 1:1 binding stoichiometry. Furthermore, the apparentequilibrium dissociation constant of the best clone (K_(D)=4 nM) is inqualitative agreement with the observed k_(diss) of 0.001 s⁻¹, assuminga k_(assoc) value about 5×10⁵ M⁻¹·s⁻¹. See Giebel, L. B., et al. (1995)Biochemistry 34(47):15430-15435, which is herein incorporated byreference.

Interesting features were observed including (1) a potential disulphidestabilized clone, (2) an extension of the consensus to very rare clones,i.e. the probability of a randomly selected clone having the seven aminoacids identical the wild-type is 1 in 5.7 billion. The data also suggestthat another around of sorting with further improve the averageaffinity. The affinity of these clones is higher than wild-type. Thehighest affinity clones obtained using only 33 pM antigen had up to 7consensus residues, and an affinity for the T7 antibody 10-fold higherthan the wild-type peptide. Thus, the present invention may be used tofurther optimize antibody peptide interactions. Binding affinities werestatistically predictable based upon the antigen concentration used forscreening. See FIG. 11. These improved T7 binding peptides may be usedas affinity tags for purification and protein detection, and improvedepitope detection.

As provided herein, to assess the functional contribution of the OmpAscaffold to high affinity binding, the 15-residue streptavidin bindingpeptide (SA-1) was genetically inserted into the yellow fluorescentprotein immediately following residue Y145. See Baird, G. S. et al.(1999) PNAS USA 96(20):11241-11246, which is herein incorporated byreference. The fluorescent protein-peptide fusion protein was expressedin soluble form in an engineered Escherichia coli strain possessing anoxidizing cytoplasm, for affinity studies. See Bessette, P. H., et al.(1999) PNAS USA 96(24):13703-13708, which is herein incorporated byreference. This fusion protein retained strong yellow fluorescencecomparable to wild-type YFP, and exhibited strong binding tostreptavidin-coated polymeric microbeads. Using flow cytometry, thedissociation rate constant of the streptavidin binding fluorescentprotein was determined to be 0.02 s⁻¹. See FIG. 12. Collectively, thesedata show that the polypeptides displayed according to the presentinvention possess high binding affinity, even in the context ofscaffolds unrelated to that used for screening.

Since peptides that include a simple consensus motif of the amino acidsHPQ have been identified in multiple phage display and mRNA displayselections against streptavidin, whether these lower affinity sequencespreviously identified using phage display would provide detectableaffinity in the display systems of the present invention was determined.To enable comparison of the phage and bacterial display peptides, abacterial display clone was constructed with the insertion,SAECHPQGPPCIEGR (SEQ ID NO:65), and the K_(D) and k_(diss) were measuredin whole cell assays. See FIG. 10A and FIG. 10B. The phagedisplay-derived peptide containing the disulfide constrained HPQ motifwas efficiently displayed on bacteria and possessed a dissociation rate(k_(diss)) 20-fold faster than that of best peptides isolated usingbacterial display (clone SA-1, FIG. 10B). In qualitative agreement withthis result, the apparent K_(D) of the cyclic HPQ clone was five-foldhigher than that of the streptavidin binding clone SA-1, confirming theimproved affinities of peptides isolated using bacterial displayrelative to those isolated using phage display.

B. OmpX Expression Vectors

An OmpX loop 2 expression vector and an OmpX loop 3 expression vectorsimilar to the OmpA loop 1 expression vector was constructed. SeeExample 2 and FIG. 6.

Table 3 provides examples of alternative insertion sites in OmpX andOmpX homologs.

TABLE 3 Sequences suitable for polypeptide display in Gram negativebacteria using E. coli OmpX (FIG. 6) and homologs in other species(preferred insertion locations in bold) Organism Loop 2 Sequence Loop 3Sequence Esherichia coli EKSRTASSGDYNKNQY KFQTTE--YPTYKNDTSD (SEQ ID NO:66) (SEQ ID NO: 72) Shigella EKSRTASSGDYNKNQY KFQTTE--YPTYKNDTSDflexneri (SEQ ID NO: 67) (SEQ ID NO: 73) Salmonella EKDRTNGAGDYNKGQYKFQTTD--YPTYKHDTSD enterica (SEQ ID NO: 68) (SEQ ID NO: 74) KlebsiellaEKDNN-SNGTYNKGQY KFQNNN--YP-HKSDMSD pneumoniae (SEQ ID NO: 69) (SEQ IDNO: 75) Serratia EKD-GSQDGFYNKAQY KFTTNA-QNGTSRHDTAD marcescens (SEQ IDNO: 70) (SEQ ID NO: 76) Yersinia pestis EKSGFGDEAVYNKAQYRFTQNESAFVGDKHSTSD (SEQ ID NO: 71) (SEQ ID NO: 77)

Suitable alternative insertion sites may be identified by multiplesequence alignment to identify non-conserved regions and are preferablychosen such that the displayed protein is located more than about 1 nMfrom the outer membrane of the cell, allowing the displayed polypeptidesto interact with arbitrary compositions of matter. See e.g. Table 3.

Other features of the OmpX expression vectors of the present inventionare similar or the same as those of the OmpA expression vector above andinclude (1) the use of the Omp signal sequence, (2) SfiI restrictionsites (3) a single resistance gene for a bacteriocidal antibiotic suchas chloramphenicol acetyltransferase, (4) a low copy origin ofreplication such as p15A for low level expression, and (5) a regulatablepromoter, such as araBAD promoter, for controlled transcription.

Likewise, the same or substantially similar experiments conducted on theOmpA expression vector described herein were conducted on the OmpXexpression vectors with similar results.

C. N/C Terminal Fusion Expression Vectors

Prior to the present invention, polypeptides were most often displayedon cell surfaces either as insertional fusions or “sandwich fusions”into outer membranes or extracellular appendages, e.g., fimbria andflagella fusion proteins or less frequently, as fusions to truncated orhybrid proteins thought to be localized on the cell surface. See Lee, etal. (2003) Trends in Biotech 23(1):45-52; Pallesen, et al., (1995)Microbiology 141:2839; and Etz, et al. (2001) J. Bacteriol.183(23):6924, which are herein incorporated by reference. Examples ofthe latter include the Lpp(OmpAaa46-159) system and the ice nucleationprotein (InP). See Georgiou, et al. (1997) Nat. Biotech. 15(1):29-34;and Shimazu, et al. (2001) Biotech. Prog. 17(1):76-80, which are hereinincorporated by reference.

The outer membrane proteins OmpA, OmpC, OmpF, FhuA, and LamB, haveenabled the display of polypeptides as relative short insertionalfusions into Omp loops exposed on the extracellular side of the outermembrane. However, the C and N-termini of these carrier proteins are notnaturally located on the cell surface which precludes the ability todisplay polypeptides as terminal fusions. As a result, proteins whichare not capable of folding in the insertional fusion context (whereintheir C and N termini are fused to the carrier protein sequence), aswell as those for which the C and N termini are physically separated inspace (e.g., single chain Fv antibody fragments) cannot be displayedeffectively as insertions. Similarly, the restriction to the use ofinsertional fusions, interferes with the display of a large number ofproteins from cDNA libraries on the cell surface.

As provided in Example 3 below, the present invention also provides anexpression vector for expressing a given polypeptide as an N-terminalfusion protein, a C-terminal fusion protein, or both, i.e., linked orfused directly to a carrier protein present on the external surface of abiological entity, and methods of making and using thereof. As usedherein, these expression vectors are referred to as “N/C terminalexpression vectors” and include the circularly permuted OmpX (CPX)expression vector exemplified in Example 3.

The N/C terminal fusion expression vectors allow longer polypeptidechains to be displayed on a surface since both termini of the displayedprotein are not constrained by insertion. The N/C terminal fusionexpression vectors of the present invention enable folding of thecarrier protein independently of the passenger polypeptide, since bothtermini are not constrained. Thus, the N/C terminal fusion expressionvectors of the present invention enable surface display of peptides andpolypeptides which require a free N or C terminus to fold efficiently,e.g. knottins, and topologically “threaded” folds. See Skerra, A. (2000)J. Mol. Recog. (13):167, which is herein incorporated by reference.

N/C terminal expression vectors of the present invention allow theenhancement of conformational diversity and surface mobility of surfaceanchored polypeptides. Specifically, the increased mobility of thepolypeptide due to its expression as a terminal fusion (as opposed to aninsertional fusion), results in a polypeptide having binding affinitiesand interactions to ligands that is substantially similar to that of thefree polypeptide, i.e., the polypeptide in solution. The presentinvention provides methods for retaining an energetically stable outermembrane protein structure that is compatible with folding, transport,and assembly to allow suitable expression of a given passenger proteinas a terminal fusion protein on the cell surface.

In some embodiments, candidate display carrier proteins, e.g., bacterialOMPs, are identified that exhibit the following properties, small (about50 kD or less, and preferably about 30 kD or less), possessextracellular loops which extend preferably 2 nM or more from thepeptidoglycan layer on the cell surface. Insertion points are chosen atthe apex of extracellular turns, preferably at sites of poor sequenceconservation (high variability) among homologs or paralogs from otherspecies. Residues in the turns of the extracellular loops inconsideration with limited phi-psi angle distributions are removed,e.g., proline. A linker is designed, see for example, FIG. 2 and FIG.13, using flexible amino acids, i.e., glycine or serine.

Using recombinant DNA techniques known in the art, an expression vectoris constructed wherein, (1) the carrier polypeptide chain is broken,preferably in the largest extracellular loop protruding maximally fromthe cell surface, e.g., Loop 2 or 3 of OmpX, (2) the naturally occurringC- and N-termini are fused using a short flexible linker sequence, suchas Gly-Gly-Ser-Gly-Gly (SEQ ID NO:78), e.g., FIG. 2 or FIG. 13, (3) aflexible linker is added by fusion to the terminus at which display isdesired, e.g., Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO:79) the desiredprotein, i.e., preceding the newly generated N-terminus for N-terminaldisplay or following the new C-terminus for C-terminal display, (4) thepassenger peptide or polypeptide (or plurality of sequences, the“library”) to be displayed is fused to the linker, e.g., FIG. 2 and FIG.4, and finally, for N-terminal display, the native signal sequence isidentified and fused to the N terminus of the polypeptide to bedisplayed. With this overall design, primers are designed to amplifygene fragments for assembly, or directly to synthesize (by total geneassembly) the designed sequence. See FIG. 13. The library of assembledgenes is digested with a suitable restriction enzyme, ligated into aregulated expression vector, e.g., pBAD18 or pBAD33, and introduced intoa host by methods known in the art such as transfection,electroporation, and the like. Plasmid DNA is prepared and multiplefrozen stocks are prepared for indefinite storage.

As provided in Example 3, sequence rearrangement of the carrier protein,in this case OmpX, was accomplished using overlap PCR, according tomethods known in the art, in order to create N/C terminal fusionexpression vectors. See FIG. 14. It should be noted that any proteinlocalized on the outer surface of a biological entity, presenting one ormore loop sequences accessible on the cell surface and the like may bemodified according to the present invention in order to generate andpresent a C-terminus, an N-terminus, or both at the outer surface of abiological entity and fused with a passenger polypeptide. Carrierproteins suitable for rearrangement for terminal fusion display from aninternal loop include outer membrane proteins, such as OmpA, OmpX, OmpT,OmpC, OmpS, LamB, TraT, IgA protease, and the like, and otherextracellular structural adhesion proteins of bacteria, such as FimH,PapA, PapG, and the like, transporter proteins of mammalian cells suchas MCAT-1, capsid and coat proteins of bacteriophage (e.g., gpVIII fromM13) and the envelope, and capsid proteins of eukaryotic cell viruses(e.g., HIV env, retroviral env, AAV capsid protein), and the like. Seee.g. Table 4. Peptide and protein insertion points were chosen to occurwithin non-conserved loop sequences. The original leader peptide or thelike was then fused to the newly generated terminus.

TABLE 4 Representative Carrier Proteins Suitable for Terminal FusionDisplay Within Internal Surface Loops Biological Entity; HomologsCarrier Protein; Examples Gram negative bacteria; (e.g. Omps; OmpA, C,F, LA, S, T, X, FepA Esherichia, Yersina, Shigella, Invasins; Inv, etc;Fimbrial & Pilus Vibrio, Pseudomonas, Proteins; FimA, FimH, PapA, PapG,F Salmonella, Enterobacter, Pilin; Flagella; FliC; S-layer protein;Klebsiella, and the like. bacteriorhodopsin; bacterial ion channels GramPositive Bacteria; (e.g. S. protein A (SpA); S-layer protein; M6Staphylococcus, protein from Streptococcus, etc. Streptococcus,Bacillus) Eukaryotic Cell Viruses Retroviral Envelope proteins; HIV,ALV/MLV, FELV Env viral capsid proteins; AAV Cap, etc. Bacterial Virus &Coat Proteins; GPIII, GPVIII (M13), 10A Bacteriophage; M13, fd, & 10B(T7 phage) T-series phage (T4, T7, and the like), lambda, and the like.Eukaryotic Cells; Yeast Cell Wall Proteins; Cwp1p, Fungal, Tip1p; Sed1p;Tir1p; YCR89W; Animal, Mamallian cell alpha helical tranporter Plant andion channel proteins; MCAT-1, MDRs; Ahesion proteins; Integrins, etc.

For N-terminal display, the peptide or protein sequence was cloned intoa multiple cloning site (MCS) following the leader peptide, preferablyimmediately following the leader peptide. The DNA sequences encoding thedisplayed peptide or protein were then fused, via PCR, to DNA sequencesencoding a mobile flexible linker of variable length, and preferablyabout 5 to about 20 amino acids. See FIG. 15 and FIG. 16. The linker Cterminus was in turn fused, using overlap PCR, to the newly generated Nterminus of the OmpX, for example, residue 54 within loop 2. Preferably,the original C and N terminus (resulting from peptidase cleavage of theleader peptide) were joined via a short flexible linker such asGly-Gly-Ser-Gly-Gly (SEQ ID NO:78), or the like, i.e. a linker whichexhibits substantially similar flexibility and conformational structureas SEQ ID NO:78. See FIG. 15. The C terminus resulting from sequencerearrangement was modified by the addition of one or more stop codons tostop translation using PCR with oligonucleotide primers incorporatingtwo stop codons, using methods known in the art.

Methods of making and using, as well as optimizing, the N/C-terminalexpression display systems include those provided above for the OmpAloop 1, OmpX-loop 2, and loop 3 expression vectors as well as thoseknown in the art.

Thus, the present invention provides expression vectors which present ordisplay polypeptides as fusion proteins to an engineered C or N terminusthat is displayed on the outer surface of a biological entity. Themethods described herein may be applied to other proteins that do notnormally present an accessible C or N terminus at the outer surface of abiological entity. This feature enables application of this invention toproteins which are optimally expressed or localized on a biologicalentity, but which may not possess a surface exposed terminus. Forexample, the Omps of bacteria, the structural proteins of bacterialfimbria, pili, and flagella, eukaryotic transporter and adhesionsproteins. See Table 4. By displaying peptides as terminal fusionproteins rather than as insertional or “sandwich” fusion proteins, thesurface displayed peptide affinity properties are more accuratelymeasured in the context of surface display. In other words, the apparentpolypeptide-target molecule binding affinity more closely approximatesvalues obtained from measurements of the same interaction in solutionwith soluble polypeptides. As a result, peptides possessing superiorperformance can be isolated and identified, and a greater variety ofprotein sequences can be displayed since one terminus of the protein isnot constrained. This approach also allows the display of two-uniquepolypeptides simultaneously at both the C and N terminus.

Terminal fusion display allows for high mobility of the surfacedisplayed molecule, increased accessibility to target molecules, andsimple proteolytic cleavage of the displayed peptide for production ofsoluble peptides. Terminal fusion display also enables theidentification of novel substrates of proteases and peptidases. See FIG.33. The N/C terminal fusion expression vectors according to the presentinvention provide a direct way for enhancing the conformationaldiversity and surface mobility of surface anchored peptides andpolypeptides. Through the increased mobility resulting from terminalfusion (as opposed to insertional fusions), the apparent affinity of apolypeptide binding to its corresponding target molecule or materialmore closely resembles that of the peptide in solution. The N/C terminaldisplay vectors allow the retention of an energetically stable outermembrane protein structure, compatible with folding, transport, andassembly for efficient display of a given passenger protein on the cellsurface.

In some embodiments, a cDNA library may be cloned into the displayposition of the N or C terminal fusion expression vector, with aterminal affinity tag, such as T7tag epitope, or a label, or the like,appended to a terminus of the cDNA clone allowing for measurement of thetotal display level on the cell surface. As used herein, the term“affinity tag” refers to a biomolecule, such as a polypeptide segment,that can be attached to a second biomolecule to provide for purificationor detection of the second biomolecule or provide sites for attachmentof the second biomolecule to a substrate. Examples of affinity tagsinclude a poly-histidine tract, protein A (Nilsson et al. (1985) EMBO J.4:1075; Nilsson et al. (1991) Methods Enzymol. 198:3, glutathione Stransferase (Smith and Johnson (1988) Gene 67:31), Glu-Glu affinity tag(Grussenmeyer et al., (1985) PNAS USA 82:7952), substance P, FLAGpeptide (Hopp et al. (1988) Biotechnology 6:1204), streptavidin bindingpeptide, or other antigenic epitope or binding domain, and the like,(Ford et al. (1991) Protein Expression and Purification 2:950), all ofwhich are herein incorporated by reference. As used herein, a “label” isa molecule or atom which can be conjugated to a biomolecule to renderthe biomolecule or form of the biomolecule, such as a conjugate,detectable or measurable. Examples of labels include chelators,photoactive agents, radioisotopes, fluorescent agents, paramagneticions, and the like.

The presence of surface localized cDNAs may be monitored using andantibody or reagent specific for the tag or label according to methodsknown in the art. Cells binding to a target protein may be then selectedusing MACS and/or FACS. The library pool may be incubated with afluorescent label of one color (such as green) and then a secondfluorescent label of a second color (such as red) to identify thepresence of a full length cDNA of interest. Clones which are red andgreen are then isolated from the library directly using cell sortingmethods known in the art.

In some embodiments, the polypeptides of an N/C terminal fusionexpression vector may be isolated or purified from the outer surface ofthe host. In other words, a polypeptide may be expressed using an N/Cterminal fusion expression vector and then produced in a soluble form(free in solution) by introducing a suppressible codon is downstream ofthe given polypeptide. Alternatively, a protease susceptible linker maybe used in place of the “suppressible” codon. The polypeptides aredisplayed on the surface at high density by induction, such as witharabinose for a period of about 2 hours. The cells are washed once ortwice in a compatible buffer, such as PBS, to remove undesired proteinsand other debris, the cells are concentrated, and a protease is added tothe cell suspension. The proteolytically cleaved polypeptide is thenharvested by removal of the bacteria by low-speed centrifugation, andtransfer of the supernatant into a fresh tube.

In some embodiments, the N/C terminal fusion expression vectors of thepresent invention can be used for the identification of substrates, suchas protease and peptidase substrates, from substrate libraries. See FIG.17. Accordingly, an N/C terminal fusion expression vector may bemodified to express a fluorescent protein using methods known in theart. For example, the use of a bicistronic expression vector comprising(1) a circularly permutated outer membrane protein, such as OmpA orOmpX, (2) a ribosomal binding site down stream of the Omp gene sequence,and (3) label such as a green fluorescent protein suitable for efficientdetection using fluorescence activated cell sorting, such as alajGFP.Expression is then monitored through the intensity of greenfluorescence.

A library of the substrates is created using methods known in the art.The substrates are fused to the N or C terminus of the N or C terminalN/C terminal fusion expression system, respectively. The substratelibrary is constructed such that a label or an affinity tag suitable forfluorescence labeling is fused to the free terminus of the passengerpolypeptide on the cell surface. See FIG. 33. The library is then grown,and cells which are green but not red are removed from the population toeliminate the isolation of false positive clones. The library is thenincubated with the enzyme (e.g., a protease or peptidase), and cellswhich loose red fluorescence while retaining green fluorescence areisolated from the population using FACS.

In some embodiments, the N/C-terminal fusion expression vectors of thepresent invention may be used to construct whole cells that can be usedas reagents. For example, one or more peptides identified using themethods herein, binding to a protein, virus, or cellular receptor, orsynthetic composition of matter, are displayed on the outer surface ofE. coli at a desired surface density. Cells can then be coupled directlyto a material, e.g., glass/silicon, gold, polymer, by virtue of peptidesselected to bind these materials, and used to capture in solutionmolecules binding to various other displayed peptides on the same cell.For optical detection, cells can co-express a fluorescent or luminescentreporter molecule such GFP, or luciferase. Flow cytometry, orfluorescence microscopy can be used to detect binding of molecularrecognition element displaying cells to the target agent, e.g., virus,cell, particle, bead, and the like. See FIG. 18 and FIG. 7.

It should be noted that although the use of bacterial proteins areexemplified herein, a variety of surface localized proteins possessingsurface exposed loops may be modified according to the present inventionto provide N/C terminal fusion expression vectors which allow thedisplay of polypeptides on the outer surface of viruses, and prokaryoticand eukaryotic cells including phage, bacteria, yeast, and mammaliancells. A variety of surface localized proteins known in the art may beused. In Escherichia coli and substantially similar species, suchproteins include OmpA, OmpX, OmpT, OmpC, OmpF, OmpN, LamB, FepA, FecA,and other beta-barrel outer membrane proteins. Proteins which exhibit atopology substantially similar to that shown in FIG. 2, i.e., presenteither a C or N terminus on the outer surface of bacteria, may also beused according to the present invention. One of ordinary skill in theart may readily identify and screen for the various surface localizedproteins that may be used in accordance with the present invention.

D. Applications of the Expression Vectors D1. Selection of Tumor orTissue/Organ Localizing Bacteria in Living Animals

As provided in Example 4, the library or a given subset of the libraryaccording to the present invention may be injected into an animal havinga xenografted tumor. After a period of time of a few minutes to severaldays, tumor or tissue targeting bacteria are isolated by removing thedesired tissues/fluids, or tumors from the organism and transferringthat sample into bacterial growth medium for bacterial amplification.

Bacterial growth, in vivo, can be monitored using a luciferase operon,autofluorescent protein expression vector, or the like. The amplifiedbacteria are then used in a substantially similar process to furtherenrich bacteria for the selected target. Host strains may be modifiedaccording to methods known in the art in order to improve selection toreduce host immune response and prevent non-specific binding in vivo.Plasmid DNA is recovered from the isolated bacteria, and the peptideencoding DNA sequence is determined. The identified peptide sequencescan then be used alone, or in combination with each other, to targetbacteria, gene therapy vectors, and other biopharmaceuticals to tumorsin humans.

D2. Immune Response Identification

The display systems of the present invention may be applied to human oranimal serum for identify dominant epitopes to which an immune responseis targeted. For example, immune responses may be quantitatively probedin both acquired and genetic diseases, e.g., autoimmune diseases,cancer, viral infections, and the like to identify disease causes,effects, and potential therapeutic intervention points.

In these embodiments, immunoglobulin (IgG) or other protein fractionsmay be purified from a test sample, such as serum, spinal fluid, orother body fluids, and labeled with biotin. This biotinylated mixture ofdifferent IgGs can then be used as antigens to select and screen forpeptides or proteins recognized by a corresponding antibody in theantigen mixture. After enrichment of the biological entity displayingantibody binding moieties, individual clones are isolated from themixture by plating, their sequences are determined by DNA sequencing.The resulting sequences would fall into distinct consensus groups thatcorrespond to different antibody specificities highly represented in themixture. See e.g. FIG. 11, FIG. 19, and FIG. 20. By performing multipleselections with the antibody mixture over a range of total antigenconcentrations, e.g., about 0.1 to about 100 nM, different consensussequences would emerge. The peptide sequence selected from the libraryisolated would, in many cases, be substantially similar or the same to acorresponding sequence present on a native protein surface. In otherwords, the display selection would allow identification of the proteinswith which the antibodies in the mixture bind to, thereby providing atarget for therapeutic intervention.

The following examples are intended to illustrate but not to limit theinvention.

Example 1 OmpA Loop 1 Expression Vector

A 15-mer random insert sequence which provides a balance betweensequence complexity and maintenance of the stability and folding andexport of OmpA was selected. See FIG. 2. It should be noted that longerlength insert, e.g., 15 mer, libraries provide more copies of shortsequences while allowing for possible longer cell binding motifsrequiring 10 or more amino acids. Although an engineered disulphidebridge may be used for stabilization, such was not used as cysteinoxidation in the E. coli periplasm could lead to aggregation and reducedexport and disulfides could potentially emerge by chance. Moreover, themembrane spanning domain of OmpA already provides a rigid structuralanchor for the peptide inserts into the more flexible loops.

After optimizing the library construction process through the use of thepBAB33L1, construction of a high quality library of about 4.5×10¹⁰independent transformants was found to be possible. This library isbelieved to be larger than any other reported bacterial display library,although a few similar sized phage libraries have been constructed. SeeVaughan, T. J., et al. (1996) Nature Biotech. 14(3):309-314, which isherein incorporated by reference. This fact is notable since librarysize has previously been shown to correlate with the quality (affinityand specificity) of the selected sequences. See Griffiths, A. D. and D.S. Tawfik (2000) Curr. Opin. Biotechnol. 11(4):338-353, which is hereinincorporated by reference. For optimal selection and screeningefficiency, expression, growth, and induction conditions, as well aspromoter strength and insert location were optimized. See FIG. 4.Importantly, a tightly-regulatable promoter was used to prevent loss ofmildly toxic sequences during growth, maintain full library diversity,and improve single round enrichment efficiency. See FIG. 5.

A. Bacterial Strains, Vectors and Plasmids

All work was performed in E. coli strain MC1061 (F⁻ araD139Δ(ara-leu)7696 galE15 galK16 Δ(lac)X74 rpsL (Str^(R)) hsdR2 (r_(K) m_(k)⁺) mcrA mcrB1), with the exception of YFP expression, which was carriedout in FA113. See Bessette, P. H., et al., (1999) PNAS USA96(24):13703-13849; and Casadaban, M. J. and S. N. Cohen, (1980) J. Mol.Biol. 138(2):179-207, which are herein incorporated by reference.Primers were obtained from Integrated DNA Technology (Coralville, Iowa),Operon-Qiagen (Valencia, Calif.), and Invitrogen (Carlsbad, Calif.).Restriction enzymes were from New England BioLabs (Beverly, Mass.).Streptavidin, R-phycoerythrin conjugate was purchased from MolecularProbes (Eugene, Oreg.). Biotinylated, and HRP conjugate, anti-T7•tagmonoclonal antibody was obtained from Novagen (Madison, Wis.).Streptavidin coated magnetic microbeads were obtained from Qiagen(Valencia, Calif.), Dynal (Brown Deer, Wis.), or Miltenyi Biotec(Auburn, Calif.). Anti-biotin mAb coated magnetic beads and anti-biotinmAb R-phycoerythrin were from Miltenyi Biotec (Auburn, Calif.).Biotinylation and fluorescent labeling with AlexaFluor488 were carriedout using the FluoReporter® Mini-biotin-XX Protein Labeling Kit andAlexa Fluor® 488 Monoclonal Antibody Labeling Kit, respectively, fromMolecular Probes (Eugene, Oreg.). Human C-reactive protein (cat# C4063)and serum albumin (cat# A3782) were from Sigma (St. Louis, Mo.).Biotinylated HIV-1 gp120 was obtained from ImmunoDiagnostics (Woburn,Mass.).

B. Vector and Library Construction

To maximize library construction efficiency, asymmetric SfiI restrictionsites were introduced into an OmpA expression vector immediatelypreceding loop 1 and following loop 4. DNA fragments containing therandom epitope insertions were synthesized by PCR, digested with SAligated into the display vector, and transformed into the E. coli strainMC1061, which can be made highly transformation competent and is ara⁻,allowing the use of the araBAD promoter for controlled OmpA expression.See Sidhu, S. S. (2000) Curr. Opin. Biotechnol. 11(6):610-616, which isherein incorporated by reference.

Plasmid pB33OmpA, contains the wild type ompA gene, including the nativeRBS, inserted downstream of the araBAD promoter in plasmid pBAD33. SeeGuzman, L., et al., (1995) J. Bacteriol. 177(14):4121-4130, which isherein incorporated by reference. It was constructed by ligation ofdigested (KpnI/HindIII) pBAD33 with a similarly digested ompA gene PCRproduct obtained using MC 1061-derived genomic DNA, and primers 1 and 2.See Table 5.

TABLE 5 Oligonucleotide primers used in polymerase chain reactions toconstruct Primer expression plasmids and libraries* 1GAGTCCAGAGGTACCAACGAGGCGCAAAAAATGAAAAAGACAGC T (SEQ ID NO: 80) 2CGTTATGTCAAGCTTTTAAGCCTGCGGCTGAGTTA (SEQ ID NO: 81) 3CAGTACCATGACACTGGCCTCATCGGCCAAAATGGTCCGACCCA T (SEQ ID NO: 82) 4AACATCGGTGACGCAGGCCAGATCGGCCAGCGTCCGGACAACGG C (SEQ ID NO: 83) 5CGTCCTGGCCTCATCGGCCAAGGATCCATGGCCTCCATGACCGGAGGACAACAAATGGGATCCGGAAATGGTCCGACCCATGAAAACC AACTGGGC (SEQ ID NO: 84) 6CGTCATCTGGCCGATCTGGCCTCCGGATCCCATTTGTTGTCCTCCGGTCATGGAGGCCATGGATCCTGCGTCACCGATGTTGTTGGTC CACTGGTA (SEQ ID NO: 85) 7CATCCGCAGGGCCCGCCGTGCATTGAAGGCCGCAATGGTCCGAC CCATGAAAAC (SEQ ID NO: 86)8 GCACGGCGGGCCCTGCGGATGGCATTCCGCGCTTTGGCCGATGA GGCCAGTGT (SEQ ID NO: 87)9 CGTCCTGGCCTCATCGGCCAA(NNS)₁₅AATGGTCCGACCCATG AAAACCAACTGGGC (SEQ IDNO: 88) 10 CGTCATCTGGCCGATCTGGCCTGCGTCACCGATGTTGTTGGTCC ACTGGTA (SEQ IDNO: 89) 11 ACTGTTTCTCCATACCCGTTTTTTTGGGCTAGCGAATTCCGTCC TGGCCTCATCGGCCAA(SEQ ID NO: 90) 12 GGCTGAAAATCTTCTCTCATCCGCCAAAACAGCCAAGCCGTCATCTGGCCGATCTGGCCT (SEQ ID NO: 91) 13 TCGCAACTCTCTACTGTTTC (SEQ ID NO: 92)14 GGCTGAAAATCTTCTCTC (SEQ ID NO: 93) 15TAGTAGCAAACGTTCTGGCAGATCTCCAAGCGTTCAATGTTGTC TCTAATTT (SEQ ID NO: 94) 16TGCCAGAACGTTTGCTACTACCTCGGGACGCTCGATGGTTCTGT TCAATTAGC (SEQ ID NO: 95)*N = A, C, G, T; S = C, G

The plasmid pB33OmpAL4 contains addition of SfiI restriction sites inthe ompA gene at positions corresponding to the beginning of the firstextracellular loop of OmpA and at the end of the fourth loop, resultingin mutations F23L, N25G, N26Q and H151G, T152Q, T155Q. PlasmidpB33OmpA14 was made via overlap PCR, using primers 1-4 (Table 5) withpB33OmpA as template. The overlap product was digested (KpnI/HindIII)and ligated to similarly digested pBAD33. Plasmids pB330T1 and pB330T4containing the T7•tag epitope inserted into loops 1 and 4, respectively,of OmpA were constructed using PCR, with pB33OmpA as template, andprimer 5 or 6 (Table 5), respectively, with primers 1 and 2 (Table 5).The overlap products were digested with SfiI and ligated with SfiIdigested pB33OmpA14. Plasmid pB330S1, containing thestreptavidin-binding peptide sequence SAECHPQGPPCIEGR (SEQ ID NO:96),inserted into OmpA loop1, was constructed by overlap PCR using primers1, 2, 7, and 8 (Table 5) with pB33OmpA14 as template. See Giebel, L. B.,et al (1995) Biochemistry 34(47):15430-15435, which is hereinincorporated by reference. Products were digested with SfiI and ligatedinto digested pB33OmpA14.

For random 15-mer library construction, primers 9 and 10 (Table 5) wereused in a PCR with pB33OmpA as the template. The resulting product waslengthened in a second PCR to enable efficient digestion, using primers11 and 12 (Table 5). The product was then digested (SfiI) and insertedinto the digested (HincII/SfiI) pB33OmpA14 vector. About 15 μg ofligated DNA was transformed to the strain MC1061 by electroporation inten aliquots. Transformed cells were pooled and incubated for 1 hour in30 ml SOC medium. Serial dilutions were plated onto LB plates with 32μg/ml chloramphenicol to determine library size. The transformed cellswere cultured in 500 ml of LB medium with 0.2% glucose and 32 μg/mlchloramphenicol and grown to an OD of 2.2. Plating of serial dilutionsof the pooled transformation mixture indicated 5×10¹⁰ independenttransformants.

The fusion protein expression plasmid encoding a yellow fluorescentprotein incorporating a peptide insertion binding to streptavidin,pB33YFP-SA, was constructed by overlap extension PCR with an AquoreaGFP-based yellow fluorescent protein gene as template with primers 13-16(Table 5), resulting in insertion of the 15 amino acid SA-1 peptide inthe permissive site between amino acids Y145 and N146 of YFP. See Baird,G. S., et al. (1999) PNAS USA 96(20):11241-11246, which is hereinincorporated by reference.

A library aliquot was placed into appropriate bacterial growth mediumcontaining more than about 0.1% glucose and propagated overnight forabout 6 to about 12 hours. The library was then diluted into freshgrowth medium at a factor of about 1:50 to about 1:100 and grown untilthe culture density (OD 600) reaches an OD value of about 0.5 to about1.0, and expression of the library elements to be display was initiatedby the addition of arabinose to the culture. The culture was thenpropagated further for about 0.1 to about 3 hours depending on thedesired surface concentration of the library element to be displayed. Inscreening random peptide libraries displayed in OmpA-L1, an inductiontime period of about 30 minutes to about 2 hours is preferred. Shorterperiods provided increased selection pressure for monovalent bindinginteractions, and consequently high affinity binding moieties.

An aliquot of the culture containing more than about 2×10¹¹ bacterialcells was then taken, washed in PBS, and resuspended in PBS at an OD ofabout 1 to about 10. The library was then mixed with one or moreligands, e.g. a protein, which has been chemically coupled to biotin,and allowed to incubated with gentle mixing, e.g., inversion or rocking,for a period of about 1 hour. The unbound ligand was then removed bywashing about 1 to about 2 times in PBS. Streptavidin coatedparamagnetic beads of about 10 nm to about 1 μm or a streptavidinconjugated fluorescent probe were then added allowing the labeled cellsto attach to the magnetic particles.

C. Magnetic Selection

Cell displaying the given polypeptide were then separated from thosethat do not by sequential application of an enrichment cycle by applyinga magnet of significant strength to the exterior face of the containerholding the library, in order to remove specifically labeled cells fromthe mixture. See FIG. 21. Cells not adhering to the magnetic particleswere then removed from the container and discarded if not of interest.The magnetic was then removed and a sterile buffer was added to thecontainer, and the cells and magnetic particles were thoroughlyresuspended using methods known in the art.

The previous two steps were then repeated about 2 to about 5 timesdepending upon the expected value of the dissociation constant of theisolated clones. For the first round of selection from a random library2 washes were sufficient unless it was known that the library containsmany sequences that bind to the target. In each successive cycle, about10 to about 1000-fold fewer cells were used for selection and the targetligand concentration (if soluble) was reduced by some factor greaterthan two, e.g., 10 fold. The ideal number of enrichment cycles isdetermined by the cycle after which no change in the number offluorescent events is observed. For example in selection for binding toHSA, the frequency of cells binding to FITC-conjugated HSA increasedafter the first round to about 0.6%, and after the second round to about10%, and then remained roughly constant at about 10% after the thirdround indicated that no further rounds of enrichment should beperformed.

After the final wash, a small volume of the sample was diluted to about1:1000 and plated onto agar plates to determine the number of clonesremaining after this enrichment cycle. The remaining volume wastransferred into a bacterial culture vessel, e.g., 250 ml culture flask,containing suitable growth medium, antibiotics, and glucose. The cellswere propagated until the reach a density of about 0.5 OD or greater,and preferable not more than about 2 hours after the cells reachstationary phase (where the culture OD is not changing). Cell were thenrelabeled with biotinylated target, and streptavidin phycoerythrin orthe like, and analyzed by flow cytometry to determine enrichment. SeeFIG. 21 and FIG. 22.

D. Flow Cytometric Screening

Flow cytometric screening of the magnetically enriched librarypopulation was used to achieve a more precise separation of only thetightest binding peptides. The enriched pool from magnetic selection wasscreened using flow cytometry for highly fluorescent cells afterincubation with a biotinylated-T7 antibody (at a final concentration of100 pM), and then streptavidin-phycoerythrin in order to assess theefficiency of selection of peptide ligands. See FIG. 21. Randomlyselected clones from the sorted population were then sequenced. See FIG.11.

For flow cytometric analysis and sorting, induced cells were typicallylabeled with biotinylated or fluorescently labeled antigen in PBS on icefor about 45 to about 60 minutes, followed by centrifugation and removalof the supernatant. When using biotinylated antigens, a secondarylabeling was carried out with 6 nM streptavidin-phycoerythrin (MolecularProbes, Eugene, Oreg.) or 1 nM anti-biotin mAb-phycoerythrin (MiltenyiBiotec, Auburn, Calif.) for 30 minutes on ice, followed bycentrifugation and removal of the supernatant. Cells were thenresuspended in cold PBS at about 10⁶ cells/ml and immediately analyzedon a Partec PAS III cytometer (Partec Inc., Muenster, Germany) equippedwith a 100 mW argon (488 nm) laser. For analysis, about 10⁴ to about 10⁶cells were interrogated, and for sorting, at least 10-fold oversamplingof the expected clonal diversity was used. Following sorting, retainedcells were either amplified for further rounds of analysis and/orsorting by growing overnight in medium containing glucose, or plateddirectly on agar for isolation of single clones. Typically, about 5 toabout 15 selected clones were confirmed for antigen binding, and theidentity of each peptide insert was determined by automated sequencingof the ompA gene contained on the isolated plasmid.

Generally, for the first round of magnetic selection, a frozen aliquotof about 2.5×10¹¹ cells was used to inoculate 500 ml of LB mediumcontaining 25 μg/ml chloramphenicol and grown at 37° C. with shaking(250 rpm) until the OD₆₀₀ was about 1 to about 1.5, at which timeL-arabinose was added to a final concentration of 0.02% (w/v). After anadditional two hours of growth, a volume corresponding to about 2.5×10¹¹cells was concentrated by centrifugation (2000×g, 4° C., 15 minutes) andresuspended in 15 ml of cold PBS.

For negative selection, 150 μl of streptavidin-coated magnetic beads(Qiagen, Valencia, Calif.) were added, and the cell/bead mixture wasincubated on ice for 30 minutes, at which time a magnet was applied tothe tube, and the unbound cells in the supernatant were removed to a newtube.

For positive selection, biotinylated antigen (about 1 to about 100 nM)was added to the supernatant fraction and incubated on ice for about 30about 60 minutes. Cells were centrifuged as above and resuspended in 7.5ml of cold PBS with 150 μl of streptavidin-coated magnetic particles(Qiagen, Valencia, Calif., or Miltenyi Biotec, Auburn, Calif.). Afterabout 30 to about 60 minutes of incubating the cells on ice withperiodic agitation, a magnet was applied to the tube, and thesupernatant was removed and discarded. The pellet was washed twice in7.5 ml of cold PBS, repelleted to the magnet each time, and finallyresuspended in LB medium and grown up overnight at 37° C. with shakingin 20 ml of LB with chloramphenicol and 0.2% glucose.

For the subsequent rounds of selection or sorting, a volume of cellscorresponding to at least 10-fold oversampling of the number of cellsretained in the previous round was subcultured to fresh LB withchloramphenicol but without glucose, grown to mid-log phase, and inducedas above. The volumes used for magnetic selection were reduced, whilemaintaining the same concentrations. In some cases, subsequent rounds ofmagnetic selection were carried out with anti-biotin mAb coated magneticparticles (Miltenyi Biotec, Auburn, Calif.).

In one round, the population was enriched to roughly 50% bindingpeptides from an initial frequency of about 1:10⁵ (about 50.000-foldenrichment). A single round of screening required only about two hoursof labor followed by overnight growth to amplify selected sequences. DNAsequencing of eight randomly chosen clones after two rounds of magneticselection revealed a strong consensus binding motif of MAPQQ (SEQ IDNO:97) or MGPQQ (SEQ ID NO:98) that conferred high affinity (K_(D)=1 nM)binding to the T7 antibody as determined using an equilibrium bindingaffinity assay. See e.g. FIG. 17. In contrast, about 12-mer to about20-mer phage display libraries rarely yield consensus sequences, likelydue to uneven amplification of selected sequences after each round ofselection. See Barry, M A., et al. (2002) VECTOR TARGETING FORTHERAPEUTIC GENE DELIVERY. Wiley-Liss; and Daugherty, et al. (1999)Protein Engineering. 12(7):613, which are herein incorporated byreference. Significantly, whole cell assays can be performed directlyusing selected clones to determine both dissociation rate constants andequilibrium affinity values of the peptide-target interaction. See FIG.11 and Daugherty, et al. (1999) Protein Engineering. 12(7):613, which isherein incorporated by reference.

The relative affinity of selected clones was rank ordered using eitherequilibrium dissociation constant measurements in the whole cell formator dissociation rate constant measurements described herein. See FIG. 13and FIG. 17.

For selection of peptides that bind to cell surface receptors whicheither do or do not become internalized, the library was mixed with apopulation of the target cells with the target cells in excess. See FIG.1 and FIG. 18. The target cells were then removed from the addedbacterial library either using immunochemical methods, chromatographicmethods, or centrifugation, and the process was repeated.

Alternatively, the library was constructed in a host cell that expressesan autofluorescent protein optimized for flow cytometric detection,e.g., alajGFP. The fluorescent protein allows cell which are eitherattached or internalized into bacteria to be detected simply by flowcytometry or fluorescence microscopy. See FIG. 18 and FIG. 21. As resultexpensive reagents are not required to detect the presence of thebinding event. The bacterial display library, exhibiting intracellularGFP is then is mixed with the target cell population, and target cellsthat exhibit green fluorescence after a short incubation of about 1minute to a few hours, and after an optional wash step are directedsorted from the population.

Bacteria were then recovered by transfer of the target cells withattached bacteria into bacterial growth medium. For selection ofinternalizing ligands, cells were treated with a drug or selectiveagent, e.g. lysozyme, which kills extracellular bacterial. Intracellularbacteria were then recovered by diluting the target cells into water tolyse the target cells, and release the bacteria. Sequential applicationof this process results in sequences which either bind to a target cell,or bind and become internalized into the target cell. See FIG. 20 andFIG. 23.

E. Protein Epitope Mapping

In many circumstances it is desirable to determine the proteins andprotein sites to which another protein binds, or to map a proteinbinding epitope. To demonstrate that the present invention may be usedfor (1) isolating protein binding peptides, and (2) determining proteinsequences to which a chosen protein binds, a protein mapping experimentwas performed as follows.

The library was first depleted of streptavidin binding peptides byincubation with streptavidin coated microbeads, e.g., from Qiagen, Inc.(Valencia, Calif.). Then the library was incubated with biotinylatedhuman C-reactive protein at 10 nM final concentration, and two rounds ofmagnetic selection were used to enrich CRP binding peptides. See FIG.22. Three rounds of MACS resulted in a population comprising more thanabout 50% binding clones using 10 nM antigen. The enriched populationwas then labeled with 100 pM CRP and cells exhibiting fluorescence abovebackground autofluorescence were sorted using FACS. One round of sortingenriched several clones exhibiting very high affinity for CRP, includingone clone, EWACNDRGFNCQLQR (SEQ ID NO:99), which was determined to be acyclic peptide with an affinity of K_(D)=1.2 nM. See FIG. 19. Twodifferent consensus sequences were obtained, a result which has veryrarely been observed using other display technologies. Equilibriumbinding affinities were measured in the whole cell format, bydetermining cell fluorescence at various concentrations of target CRP.See e.g. FIG. 17. CRP binding clones are likely to be useful asinexpensive diagnostic reagents.

F. Selection of High Affinity Protein Binding Peptides Using KineticSelection

The library was incubated with a 1:1 mixture of streptavidin coatednano-spheres (50 nM) and streptavidin coated microparticles (Qiagen,Valencia, Calif.). Magnetic selection was used to separate bindingclones from non-binding clones. Two rounds of selection provided apopulation of more than about 25% streptavidin binding cells. Theenriched population was the labeled with streptavidin at 1 nMconcentration, washed 1× in PBS, and the resuspended in PBS with 100 μMbiotin as a competitor. This process step is used to favor clones withslow dissociation rate constants. After 1 hour, cells retainingdetectable fluorescence were sorted using FACS. Individual clones wereisolated by plating on agar plates and picking colonies after overnightgrowth. Clones from both magnetic selection and magnetic selection+FACSwere sequenced, and their dissociation rate constants were measuredusing flow cytometry. See FIG. 24. The dissociation rate, andequilibrium dissociation constants were measured using flow cytometry.See FIG. 13 and FIG. 17. The highest affinity clone had an affinity of 4nM and a dissociation rate constant of 0.0007 s⁻¹. The sequence functiondata can be used to establish sequence function relationships.

G. Selection of HSA and gp120 Binding Peptides

The above process was applied to isolated peptides that bind to HIV-1gp120 (as potential viral entry inhibitors) and to human scrum albuminfor determining feasibility of drug delivery and purificationapplications. See Sato, A. K., et al. (2002) Biotechnol. Prog.18(2):182-192, which is herein incorporated by reference. Examples ofselected peptides are shown in FIG. 25 and FIG. 26. The affinities ofpeptides isolated using the methods of the present invention are foundto be significantly higher the affinities of peptides isolated usingphage display for identical targets. See Table 6.

TABLE 6 Equilibrium dissociation constants for peptides selected fromTarget Protein K_(D) (nM) T7 MAb 0.3 C-RP 1.2 SA 4 HSA 100 GP120 2

The present invention allows the identification of optimal cysteinplacements to form disulphide constrained loops conferring high bindingaffinity without explicit library design, thereby alleviating the needto construct and screen ten or more different libraries, and removingcritical assumptions that have limited the affinities of isolatedligands in earlier studies. See e.g. Giebel, L. B., et al. (1995)Biochemistry 34(47):15430-15435, which is herein incorporated byreference. For example, selections for binding to streptavidin yielded astrong preference for CX₃C ligands in all rounds of selection. Thoughseveral reports have previously described the screening of both linearand disulphide constrained peptide libraries (with differing lengths),the generation and screening of a CX₃C type library using any reporteddisplay technology has not been described previously.

Since loop rigidity has been shown to correlate with binding affinity,the additional rigidity imparted by the more tightly constrained loopappears to benefit affinity. It appears likely that phage displayselections using “built-in” three-residue turns might yield affinityimprovements relative to previously selections. While the results withstreptavidin were expected, peptides containing putative disulphideloops were present in peptides binding to each of the target ligandstested (T7 antibody, HSA, gp120, and CRP) despite a 1000-fold reducedprobability of occurrence. While a strong consensus sequence of IXNXRGF(SEQ ID NO:100) was present in clones from the selection for CRPbinding, FACS screening of the enriched pool result in the isolation ofa peptide having the consensus and being flanked by two cysteinesCNDRGFNC (SEQ ID NO:101), i.e., 6 residue loop. Several such peptidesdeviated to different extents from the consensus suggesting that thepresence of a disulphide compensated for other deviations from theconsensus. See FIG. 11, FIG. 19, and FIG. 25.

H. Clonal Affinity Characterization

To obtain equilibrium binding curves, cells were labeled over a range ofconcentrations, e.g., about 0.1 to about 200 nM, of fluorescentlyconjugated target proteins (streptavidin-phycoerythrin orCRP-AlexaFluor488) and analyzed by flow cytometry, as above. Thecorresponding mean fluorescence versus concentration data were fit to amonovalent binding isotherm to obtain the apparent K_(D). Dissociationrates of streptavidin-binding clones were measured in the presence ofabout 1 to about 2 μM biotin. Cells were labeled with 50 nMstreptavidin-phycoerythrin for 30 minutes at room temperature. The cellswere then pelleted, resuspended in PBS with biotin, and immediatelyanalyzed by flow cytometry. Fluorescence data were collectedcontinuously for about five minutes. The dissociation rate constantswere then determined as described previously. See Daugherty, P. S., etal. (1998) Protein Eng. 11(9):825-832, which is herein incorporated byreference.

For analysis of peptide affinity in a soluble scaffold,streptavidin-binding peptide SA-1 fused within a loop of YFP wasprepared by cytoplasmic expression in E. coli strain FA113, inducedovernight at room temperature. The soluble protein was isolated usingB-PER II bacterial protein extraction reagent (Pierce Biotechnology,Rockford, Ill.) following the manufacturer's protocol. About 10⁷streptavidin coated magnetic beads (Dynal Inc., Brown Deer, Wis.) wereadded to 40 μl of cell lysate and equilibrated at room temperature for20 minutes. The beads were washed once in 2 ml of PBS; biotin was addedto a final concentration of 1 μM, and immediately analyzed by flowcytometry as above. Lysate from a strain expressing YFP with a T7•taginsertion at the same location was used as a negative control.

Example 2 OmpX Loop 2 and OmpX Loop 3 Expression Vectors

While the following protocol specifically describes the construction ofvectors for the display of polypeptides and polypeptide libraries inloop 2 of OmpX, this procedure may be readily applied to loop 3 of OmpX,by consideration of the non-conserved regions in loop 3 as described inTable 3, by one skilled in the art. In loop 3, peptide insertions arepreferred between residues 94-99, and preferably between residues 95-97,with Pro96 removed. The wild-type OmpX gene from E. coli MC 1061:

(SEQ ID NO: 102) atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagctgcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagc/tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ccg/acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttctaataawas amplified using primer 1 and primer 2 introducing a KpnI cut site atthe front of the gene and SfiI and HindIII cut sites at the end of thegene and inserted into pBAD33 using KpnI and HindIII digestions tocreate pB33OmpX. Table 7 shows the primers used.

TABLE 7 Primer Description Primer Sequence 1 OmpX forwardttcgagctcggtacctttgaggtggttatgaaaaaaattg w/KpnI (SEQ ID NO: 103) 2 OmpXreverse aaaacagccaagcttggccaccttggccttattagaagcg w/SfiI, gtaaccaacaccHindIII (SEQ ID NO: 104) 3 OmpX SfiI,gcgagcatgaccggcggccagcagatgggtggcgggagtt T7tag loop2ctggtgactacaacaaaaac forward (SEQ ID NO: 105) 4 OmpX SfiI,ctggccgccggtcatgctcgccatttggcccgactggccg T7tag loop2cttgcagtacggcttttctc reverse (SEQ ID NO: 106) 5 Making OmpXagaaaagccgtactgcaagcggcgggagttctggtgacta template (SEQ ID NO: 107) 6Ompx reverse tatctaagcttttattagaagcggtaaccaacacc w/HindIII (SEQ ID NO:108) 7 OmpX 3C aagcaagctgcaagtccgaagcggccagtcgggccaanns librarynnsnnsnnstgcnnsnnsnnstgcnnsnnsnnsnnsggcg ggagttctggtgacta (SEQ ID NO:109) 8 OmpX alpha tgcaagtccgaagcggccagtcgggccaannstgctgcnn CT librarysnnsnnsnnstgcnnsnnsnnsnnsnnsnnsnnstgcnns ggcgggagttctggtgacta (SEQ IDNO: 110)Primer 1/primer 3 and primer 4/primer 2 were used in separate PCRreactions with pB33OmpX as the template to produce fragments that wereused in an overlap extension PCR.

The final product includes a SfiI site before a T7tag peptide epitopewith four flanking residues on either side inserted within loop 2 ofOmpX, resulting in a S74G substitution. The product was then digestedwith KpnI and HindIII and ligated to similarly digested pBAD33 to createpB33OmpX-T2. The pB33OmpX-T2 plasmid was then cut with SfiI to createthe vector that was used to generate the OmpX libraries. The plasmidpB33OmpX-temp was created lacking the SfiI restriction sites and the T7epitope that was used as the template for the PCR to generate thelibrary insert. pB33OmpX-temp was made using PCR, using primer 5 andprimer 6 to create a “megaprimer” with pB33OmpX-T2 as template. Themegaprimer and primer 1 were then used in a PCR reaction withpB33OmpX-T2 as template. The product was digested with KpnI and HindIIIand ligated to similarly digested pBAD33. Primer 7 and Primer 8 wereused separately as the forward primers to create the various libraryinserts with primer 2 as the reverse primer and pB33OmpX-temp as thetemplate. The product was digested with SfiI and ligated to similarlycut pBAD33OmpX-T2 to generate the OmpX display libraries.

Example 3 Circularly Permuted OmpX (CPX)

Display and expression of passenger polypeptides as N or C terminalfusions is accomplished by topological permutation of an Omp as shown inFIG. 14. Sequence rearrangement of an outer membrane protein, in thiscase OmpX, was accomplished using an overlap extension PCR methods knownin the art in order to create either N or C terminal fusion constructs.See Ho, et al. (1989) Gene 77(1):51-59, which is herein incorporated byreference; and see FIG. 14, FIG. 27, FIG. 28, FIG. 30, and FIG. 32.Polypeptide passenger insertion points are chosen to occur withinnon-conserved, surface exposed loop sequences of surface exposedproteins, such as monomeric Omps (including OmpA, OmpX, OmpT, and thelike) using methods known in the art.

The DNA sequence of the N/C terminal fusion expression vector providesthe following contiguous components fused or linked in linear order fromN to C terminus (See FIG. 14):

-   -   1. A DNA sequence encoding an N-terminal leader peptide, such as        the native N-terminal leader peptide from an outermembrane        localized protein (e.g., OmpX, OmpA, or the like.    -   2. A DNA restriction enzyme cleavage site (for efficient library        construction),    -   3. A DNA sequence encoding given polypeptide to be expressed and        displayed on the cell surface,    -   4. A DNA sequence encoding peptide linker, which may include        entities commonly employed in the recombinant DNA and protein        engineering arts, such as a proteolytic cleavage site that        allows peptide release from the cell surface, and the like,    -   5. A carrier protein sequence beginning with the amino acid        downstream, preferably immediately downstream, of the insertion        point at which display is desired (e.g., wt OmpX aa 54) and        ending with carrier's native-terminus excluding native stop        codon(s).    -   6. A DNA sequence encoding a short, flexible peptide linker        sequence (e.g., GGSGG (SEQ ID NO:78), or others known in the        art),    -   7. A DNA sequence encoding the carrier protein's sequence        beginning with the amino acid upstream, preferably immediately        upstream, of the carrier's native leader peptide and ending with        the amino acid upstream, preferably immediately upstream, of the        chosen insertion site, and    -   8. Two stop codons for efficient termination followed by        appropriate restriction enzyme cleavage sites (e.g., SW, or the        like).

Terminal Fusion Expression Vectors

The following sequences and primers were used to construct the N/Cterminal fusion expression vectors, and the resulting DNA sequencesaccording to the specifications of FIG. 14 using methods known in theart:

Protein sequence of wild-type E. coli pro-OmpX (pre-signal peptidecleavage): (SEQ ID NO: 111)(SS)MKKIACLSALAAVLAFTAGTSVAATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTEKSRTAS/SGDYNKNQYYGITAGPAYRINDWASIYGVVGVGYGKFQTTEY/P/TYKHDTSDYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWIAGVGYRF** DNA sequence of wild-type E. coli OmpX:(SEQ ID NO: 112) atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagctgcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagc/tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ccg/acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttctaataaA. Contiguous (fused) DNA Sequences for the Display of the T7Tag PeptideEpitope as an N-Terminal Fusion within OmpX Loop 2 (Between Amino Acids53 and 54 of Mature OmpX):

(SEQ ID NO: 113) (SS)atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct-gcgacttctact (T7tag): (SEQ ID NO: 114)atggcgagcatgaccggcggccagcagatgggt (Linker): (SEQ ID NO: 115)ggaggccagtctggccag

OmpX amino acids 54 to end (STOP): (SEQ ID NO: 116)tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatacccgacctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttc Peptide Linker: (SEQ ID NO: 117)ggaggaagcgga OmpX aa 1 (first residue of structure)-53: (SEQ ID NO: 118)gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgt actgcaagc Stopcodons: (SEQ ID NO: 119) taataaProtein Sequence Resulting from Translation of the Above DNASequence=OmpX Signal Sequence/T7/SfiI/AA54/AA148/AA1/AA53:

(SEQ ID NO: 120) MKKIACLSALAAVLAFTAGTSVA/MASMTGGQQMG/G/GQSGQ/SGDYNKNQYYGITAGPAYRINDWASIYGVVGVGYGKFQTTEYPTYKHDTSDYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWIAGVGYRF/GGSG/ATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTEKSRTAS**B. Contiguous DNA Sequences for the Display of the T7 Epitope as aC-Terminal Fusion within OmpX Loop 3 (Between Amino Acids 95/97)

The order of the genetic elements encoding the C-terminal Loop 3 displayvector is: signal sequence/OmpX 97-148/Linker/OmpX 1-95/Linker/T7tagpeptide/stop codons: This fusion protein is encoded by the DNA sequence:

(SEQ ID NO: 121) atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct/acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttc/ggaggaagcgga/gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagctctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ggaggaagcggaggaa/tggcgagcatgaccggcggccagcagatgggt/taataaProtein Sequence Resulting from Translation of the DNA SequenceImmediately Above=SignalPeptide/AA97-AA148/Linker/AA1-AA95/Linker/T7Tag:

(SEQ ID NO: 122) MKKIACLSALAAVLAFTAGTSVA/TYKHDTSDYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWIAGVGYRF/GGSG/ATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTEKSRTASSGDYNKNQYYGITAGPAYRINDWASIYGVVGVGYGKFQTTEY/GGSGGMASMTGGQQMG**

T7tag peptide encoding sequence: (SEQ ID NO: 123)5′ atggcgagcatgaccggcggccagcagatgggt (SEQ ID NO: 124) MASMTGGQQMGStreptavidin binding peptide encoding sequence: (SEQ ID NO: 125)5′ accgtgctgatttgcatgaacatctgttggacgggcgaaactcag (SEQ ID NO: 126)TVLICMNICWTGETQ SacI and KpnI 5′ sites: (SEQ ID NO: 127)ttcgagctcggtacctttgaggtggtt Signal sequence: (SEQ ID NO: 128)atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcac cgcaggtacttccgtagct(SEQ ID NO: 129) MKKIACLSALAAVLAFTAGTSVA SfiI & Hind III 3′ sites: (SEQID NO: 130) ggccaaggtggccaagcttggctgtttt

C. Display of Peptides Binding to Streptavidin, T7-Tag MonoclonalAntibody, and C-Reactive Protein as N-Terminal Fusion Proteins

To construct the N-terminal T7tag display vector, primers 1-14:

Primer (5′→3′) 1: Length 60 Melting Tm 48 Sense strand (SEQ ID NO: 131)ttcgagctcggtacctttgaggtggttatgaaaaaaattgcatgtctttc agcactggcc Primer(5′→3′) 2: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 132)tttcagcagtggccgcagttctggctttcaccgcaggtacttccgtagct atggcgagca Primer(5′→3′) 3: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 133)agctatggcgagcatgaccggcggccagcagatgggtggaggaagcggag gatctggtga Primer(5′→3′) 4: Length 60 Melting Tm 50 Sense strand (SEQ ID NO: 134)cggaggatctggtgactacaacaaaaaccagtactacggcatcactgctg gtccggctta Primer(5′→3′) 5: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 135)gctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagt gggtgtgggt Primer(5′→3′) 6: Length 60 Melting Tm 50 Sense strand (SEQ ID NO: 136)gtagtgggtgtgggttatggtaaattccagaccactgaatacccgaccta caaacacgac Primer(5′→3′) 7: Length 60 Melting Tm 51 Sense strand (SEQ ID NO: 137)cgacctacaaacacgacaccagcgactacggtttctcctacggtgcgggt ctgcagttca Primer(5′→3′) 8: Length 60 Melting Tm 48 Sense strand (SEQ ID NO: 138)cgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttac gagcagagcc Primer(5′→3′) 9: Length 60 Melting Tm 50 Sense strand (SEQ ID NO: 139)cttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgcc ggtgttggtt Primer(5′→3′) 10: Length 60 Melting Tm 48 Sense strand (SEQ ID NO: 140)tgccggtgttggttaccgcttcggaggaagcggagcgacttctactgtaa ctggcggtta Primer(5′→3′) 11: Length 60 Melting Tm 48 Sense strand (SEQ ID NO: 141)ctgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaa atgggcggtt Primer(5′→3′) 12: Length 60 Melting Tm 51 Sense strand (SEQ ID NO: 142)acaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagc ccgctgggtg Primer(5′→3′) 13: Length 60 Melting Tm 49 Sense strand (SEQ ID NO: 143)gcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtact gcaagctaat Primer(5′→3′) 14: Length 47 Melting Tm 49 Antisense strand (SEQ ID NO: 144)aaaacagccaagcttggccaccttggccttattagcttgcagtacggand the numbering scheme corresponding to FIG. 28 and FIG. 29, were usedin standard PCR using methods known in the art to give the followingsequences:

5′ flank & Signal sequence: (SEQ ID NO: 145)/ttcgagctcggtacctttgaggtggtt/atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct/ nt1-159: (SEQ ID NO:146) /gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccg tactgcaagc/nt160-285: (SEQ ID NO: 147)tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ccg/ nt289-441: (SEQ ID NO: 148)acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttac cgcttc/taataa

The above PCR fragments are then fused using overlap extension PCRreactions using primers 1-14 according to the scheme of FIG. 25,resulting in the full length N-terminal T7tag display vector encoded bythe following:

(SEQ ID NO: 149) 5′ ttcgagctcggtacctttgaggtggtt/atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct/gcgacttctact/atggcgagcatgaccggcggccagcagatgggt/ggaggccagtctggccag/tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ccg/acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttc/ggaggaagcgga/gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagc/taataa 3′

D. To Construct the N-Terminal Loop 2 According to FIGS. 28 and 29 andC-Terminal Loop-3 Display Vectors According to FIG. 30 and FIG. 31 theFollowing DNA Sequences:

5′ flanking & Signal sequences (Prime w/PSD 515): (SEQ ID NO: 150)ttcgagctcggtacctttgaggtggtt/atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct nt1-159: (SEQ ID NO:151) gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgt actgcaagc nt160-285:(SEQ ID NO: 152) tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac nt289-441: (SEQ ID NO: 153)acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttac cgcttctaataawere synthesized, and overlapped using PCR using the following primers1-16:

Primer (5′→3′) 1: Length 45 Melting Tm 49 Sense strand: (SEQ ID NO: 154)ttcgagctcggtacctttgaggtggttatgaaaaaaattgcatgt Primer (5′→3′) 2: (SEQ IDNO: 155) Length 57 Melting Tm 48 Antisense strand:gcggtgaaagccagaactgcggccagtgctgaaagacatgcaattttttt cataacc Primer(5′→3′) 3: Length 57 Melting Tm 48 Sense strand: (SEQ ID NO: 156)tggctttcaccgcaggtacttccgtagctacctacaaacacgacaccagc gactacg Primer(5′→3′) 4: Length 57 Melting Tm 49 Antisense strand: (SEQ ID NO: 157)ttttccatcgggttgaactgcagacccgcaecgtaggagaaaccgtagtc gctggtg Primer(5′→3′) 5: Length 57 Melting Tm 48 Sense strand: (SEQ ID NO: 158)ttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccg tattcgt Primer(5′→3′) 6: Length 57 Melting Tm 50 Antisense strand: (SEQ ID NO: 159)gcggtaaccaacaccggcaatccaggtgcctacgtcaacgctacgaatac ggctctg Primer(5′→3′) 7: Length 57 Melting Tm 48 Sense strand: (SEQ ID NO: 160)ggtgttggttaccgcttcggaggaagcggagcgacttctactgtaactgg cggttac Primer(5′→3′) 8: Length 57 Melting Tm 48 Antisense strand: (SEQ ID NO: 161)ccgcccattttgttcatttggccctgagcgtcgctctgtgcgtaaccgcc agttaca Primer(5′→3′) 9: Length 57 Melting Tm 49 Sense strand: (SEQ ID NO: 162)gaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaaca gcccgct Primer(5′→3′) 10: Length 57 Melting Tm 51 Antisense strand: (SEQ ID NO: 163)cagtacggcttttctcggtgtaagtgaaagaaccgatcacacccagcggg ctgttgt Primer(5′→3′) 11: Length 57 Melting Tm 49 Sense strand: (SEQ ID NO: 164)cgagaaaagccgtactgcaagctctggtgactacaacaaaaaccagtact acggcat Primer(5′→3′) 12: Length 57 Melting Tm 51 Antisense strand: (SEQ ID NO: 165)tgcttgcccagtcgttaatgcggtaagccggaccagcagtgatgccgtag tactggt Primer(5′→3′) 13: Length 57 Melting Tm 49 Sense strand: (SEQ ID NO: 166)cgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattcc agaccac Primer(5′→3′) 14: Length 57 Melting Tm 48 Antisense strand: (SEQ ID NO: 167)ccggtcatgctcgccattcctccgcttcctccgtattcagtggtctggaa tttacca Primer(5′→3′) 15: Length 57 Melting Tm 49 Sense strand: (SEQ ID NO: 168)cgagcatgaccggcggccagcagatgggttaataaggccaaggtggccaa gcttggc Primer(5′→3′) 16: Length 19 Melting Tm 49 Antisense strand: (SEQ ID NO: 169)aaaacagccaagcttggccaccording to the scheme of FIG. 30, resulting in the full lengthC-terminal display vector encoded by:

(SEQ ID NO: 170) ttcgagctcggtacctttgaggtggttatgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagct/acctacaaacacgacaccagcgactacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttc/ggaggaagcgga/gcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagc/tctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatac/ggaggaagcggagga/atggcgagcatgaccggcggccagcagatg ggt/taataa

E. Construction of an OmpX Display Scaffold Utilizing Only 19 of the 20Standard Amino Acids, i.e., No Leucine Codons

Plasmid pB33NLXT2 (No Leucine OmpX with T7tag in loop 2) was isolatedfrom a no leucine OmpX library (NLL) constructed in plasmid expressionvector pBAD33OmpX-T7tag-L2, which encodes OmpX with the T7tag peptideinserted into Loop 2, under the transcriptional control of the arabinosepromoter (B), on a low-copy plasmid possessing a p15A origin ofreplication. by selecting with FACS for T7tag display in a leucineauxotroph (MC1061) grown in minimal medium lacking leucine. This OmpXvariant contains the mutations L17V, L14V, L10V, L26V, L371, L113V,L123V, wherein the amino acid numbering is based on the mature form ofwild type OmpX.

The “no leucine” library used above, allowing valine or isoleucine ateach leucine codon, was constructed by performing overlap extension PCR,using methods known in the art. Plasmid pB33XT2 was used as a templatefor three separate reactions with primers PD674/675, PD676/677, andPD678/180. See Table 8.

TABLE 8 primer sequence PD179 tcgcaactctctactgtttc (SEQ ID NO: 171)PD180 ggctgaaaatcttctctc (SEQ ID NO: 172) PD515ttcgagctcggtacctttgaggtggttatgaaaaaaattg (SEQ ID NO: 173) PD632cagtagaagtcgctccgcttcctccgaagcggtaaccaacaccg g (SEQ ID NO: 174) PD633ggaggaagcggagcgacttctactgtaactggcggttacgcaca g (SEQ ID NO: 175) PD634aaaacagccaagcttggccaccttggccttattagcttgcagta cggcttttctcg (SEQ ID NO:176) PD674 gttatgaaaaaaattgcatgtrtttcagcarttgccgcagttrt tgctttcaccgcaggt(SEQ ID NO: 177) PD675 tgttgtcttcttcatagcggtatttaaygttgaaaccgcccatt ttgt(SEQ ID NO: 178) PD676 ccgctatgaagaagacaacagcccgrttggtgtgatcggttctt tcac(SEQ ID NO: 179) PD677 aacgttttccatcgggttgaactgaayacccgcaccgtaggaga aac(SEQ ID NO: 180) PD678 ttcaacccgatggaaaacgttgctrttgacttctcttacgagca gag(SEQ ID NO: 181) PD703 ctgcccagactgccctccctggccagactggccagctacggaagtacctgc (SEQ ID NO: 182) PD704 ggagggcagtctgggcagtctggtgactacaacaaa (SEQID NO: 183) PD707 ctgactgaggccagtctggccagnnsnnstgcnnsnnsnnsnnsnnsnnsnnstgcnnsnnsggagggcagtctgggcag (SEQ ID NO: 184) PD753gctttcaccgcaggtacttctgactgaggccagtctggcc (SEQ ID NO: 185)

The resulting products were purified, pooled, and amplified in a secondround with primers PD515/180. The product was digested with KpnI/HindIII(as well as DpnI and PstI to remove template carryover), repurified, andligated to the large fragment of pBAD33 that had been digested withKpnI/HindIII.

Plasmid pB33NLCPX (No Leucine Circularly Permuted OmpX in pBAD33) wasconstructed by PCR amplification of pB33NLXT2 in three reactions withPD515/703, PD704/632, and PD633/634. The fragments generated were eachpurified and pooled in a second round overlap reaction with outsideprimers PD515/634. The resulting product was then purified, digestedwith KpnI/HindIII (as well as DpnI to remove template carryover),repurified, and ligated to the large fragment of pBad33 that had beendigested with KpnI/HindIII.

F. Construction of an N-Terminal Peptide Library within Loop 2 of NLCPX.

The NLCPX-C7C library was constructed by PCR amplification of pB33NLCPXwith primers PD707/180. The product was diluted 25-fold into a fresh PCRwith primers PD753/180, in order to extend the length of the fragment onthe 5′ end. The resulting product was purified, digested with SfiI,repurified, and ligated to the large fragment resulting from digestionof pB33NLCPX with SfiI/HincII. The ligation mixture is then transformedinto electrocompetent E. coli MC 1061 cells using electroporation, andcells are grown overnight in LB supplemented with glucose, resulting inthe N-terminal peptide display library which can be aliquoted or furtheramplified by growth.

G. Non-Canonical Amino Acid Analogs

Non-canonical amino acid analogs which can be recognized andincorporated by the native or engineered cellular translationalmachinery, can be displayed more efficiently by redesigning thescaffolds described herein as follows. See Link, A. J. et al. (2003)Curr. Opin. Biotechnol. 14(6):604, which is herein incorporated byreference. All codons corresponding to one or more native amino acidsare removed by first constructing an Omp gene variant library via geneassembly mutagenesis in which all of the codons in are randomized togenerate codons that encode alternative amino acids. See Bessette, etal. (2003) Methods. Mol. Biol. 231:29-37, which is herein incorporatedby reference. Selection or screening is then used to isolate Ompvariants that efficiently display a passenger protein in the absence ofthe corresponding natural amino acid.

For example, to create a scaffold that efficiently displays the leucineanalog trifluorleucine, all leucine codons were randomized such thatthey could encode valine or isoleucine at each position. This librarywas sorted by FACS for T7tag display in medium comprising 19 standardamino acids (no leucine) supplemented with trifluoroleucine. One of theresulting clones (NLOmpX T7tag) exhibits T7tag display in media lackingleucine at a level equivalent to media with 20 amino acids. In contrast,the wild-type OmpX scaffold exhibits a substantially reduced level ofdisplay of the epitope in 19 amino acids. This mutant OmpX sequence(NLOmpX) has all leucine codons replaced with valine, except at position37 of the mature protein, which is replaced with isoleucine. Using theNLOmpX as a starting point for creating and screening a library allowsfor the ability to perform negative selections in media lacking leucine,in order to remove binders that do not contain leucine codons.

A scaffold deficient in at least one of the 20 standard amino acids,e.g., is preferred for screening libraries that incorporated analogs ofthe deficient amino acid. The reason is that with too many leucinecodons in the OmpX DNA sequence, the removal of leucine, and addition ofa leucine analog, such as trifluorleucine, inhibits the expression ofthe carrier OmpX. See FIG. 34. Thus, wild-type OmpX can not be madewithout Leu in the medium, but with leucine present, the leucine analogcan not be incorporated since the rates of incorporation are different.Therefore, removing the leucines from the scaffold (OmpX) allowsscaffold synthesis without any leucine present. As a result, one mayreadily screen for polypeptide libraries that incorporate leucineanalogs.

Example 4 Assay Using the Expression Vectors

The following two strategies were used to isolate sequences which bindto tumor cells, and potentially internalize. First, the bacterialdisplay library was selected for binding by incubation with tumor cells,and selective sedimentation of tumor cells. A single round of enrichmentby sedimentation was used to enrich binding or internalizing sequences.Two additional rounds were performed incorporating a step designed toselectively kill extracellular bacteria with the antibiotic gentamycin.Intracellular bacteria were then recovered by osmotic shock conditionsresulting in preferential tumor cell lysis. The two rounds of selectionincorporating a gentamycin selection steps resulted in a furtherincrease in the percentage of green tumor cells in the FACS invasionassay. See FIG. 23. After the first three rounds of enrichment by simpleco-sedimentation of bacteria adhering to tumor cells and gentamycinselection, a GFP expression vector was electroporated into each of thelibrary pools resulting from each round to monitor selection success theremaining library population to facilitate quantitative and efficientFACS screening. See FIG. 23. Two rounds of FACS screening providedadditional enrichment. See FIG. 20. After five rounds of selection forinternalization into ZR-75-1 tumor cell line, (ATCC No. CRL-1500) fromATCC (Manassas, Va.), single clones were isolated and assayed for theirinternalization efficiency, as suggested by the gentamycin protectionassay. The isolated clones exhibited up to about a 200-fold (0.005→1.0%)increased ability to internalize into the target cell lines, relative tonegative controls. The sequences of a panel of isolated sequences fromround 5 are presented in FIG. 7.

To demonstrate that peptides selected by bacterial display boundspecifically to tumor cells, bacterial cells displaying tumor targetingpeptides, and expressing an autofluorescent protein, e.g., EGFP, wereincubated with tumor cells for one hour. Non-bound cells were washedfrom the tumor cell surfaces and images were acquired using fluorescencemicroscopy. See FIG. 14. Tumor cells incubated with OmpA displayingbacteria only, were non-fluorescent (FIG. 14), while tumor targetingpeptide displaying bacteria bound specifically to tumor cell clumps(ZR-75-1). Therefore, fluorescent protein expressing, peptide displayingbacteria can be used as an infinitely renewable diagnostic reagents in avariety of assay platforms known to one skilled in the art, such asELISA, fluorescence microscopy, and flow cytometry.

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

1.-20. (canceled)
 21. A composition comprising: a replicable biologicalentity; and a passenger polypeptide displayed on the surface of thereplicable biological entity, wherein the passenger polypeptide is fusedto an extracellular terminus of a circularly permuted bacterial outermembrane protein.
 22. The composition of claim 21, wherein the bacterialouter membrane protein is OmpA.
 23. The composition of claim 21, whereinthe bacterial outer membrane protein is OmpX.
 24. The composition ofclaim 21, wherein the passenger polypeptide is a substrate for anenzyme.
 25. The composition of claim 21, wherein the passengerpolypeptide comprises a label.
 26. The composition of claim 25, whereinthe label is a fluorescent agent.
 27. The composition of claim 25,wherein the label is a C-terminal label.
 28. The composition of claim25, wherein the label is an N-terminal label.
 29. The composition ofclaim 21, wherein the C-terminus of the circularly permuted bacterialouter membrane protein is exposed on the surface of the replicablebiological entity.
 30. The composition of claim 21, wherein theN-terminus of the circularly permuted bacterial outer membrane proteinis exposed on the surface of the replicable biological entity.
 31. Thecomposition of claim 21, wherein the replicable biological entity is abacterial cell, a yeast cell or a mammalian cell.
 32. The composition ofclaim 21, wherein the replicable biological entity is a bacterial cell.33. The composition of claim 32, wherein the bacterial cell isEscherichia coli.
 34. A polypeptide display library comprising: aplurality of replicable biological entities, each member of theplurality comprising a passenger polypeptide displayed on the surface ofthe member replicable biological entity, wherein the passengerpolypeptide is fused to an extracellular terminus of a circularlypermuted bacterial outer membrane protein.
 35. The polypeptide displaylibrary of claim 34, wherein the bacterial outer membrane protein isOmpA.
 36. The polypeptide display library of claim 34, wherein thebacterial outer membrane protein is OmpX.
 37. The polypeptide displaylibrary of claim 34, wherein the passenger polypeptide comprises alabel.
 38. The polypeptide display library of claim 37, wherein thelabel is a fluorescent agent.
 39. The polypeptide display library ofclaim 37, wherein the label is a C-terminal label.
 40. The polypeptidedisplay library of claim 37, wherein the label is an N-terminal label.41. The polypeptide display library of claim 34, wherein the replicablebiological entity is a bacterial cell, a yeast cell or a mammalian cell.42. The polypeptide display library of claim 34, wherein the replicablebiological entity is a bacterial cell.
 43. The polypeptide displaylibrary of claim 42, wherein the bacterial cell is Escherichia coli.